Published online before print June 30, 2006
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,
,1
* Inflammation Program and Departments of
Microbiology and
Internal Medicine, University of Iowa and the VA Medical Center, Iowa City
1 Correspondence: Inflammation Program, University of Iowa, 2501 Crosspark Rd., MTF D154, Coralville, IA 52241. E-mail: lee-ann-allen{at}uiowa.edu
 TOP ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Key Words: phagocytosis • complement • live-vaccine strain • MDM • tularemia • lectinophagocytosis
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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A key element of pathogenesis is the ability of Ft to evade killing by macrophages [3 ]. Although we know little about this infection at the molecular level, published data indicate that Ft phagosomes resist fusion with lysosomes [4 ], and late in infection, bacteria escape from the phagosome and replicate in the macrophage cytosol [5 6 7 ]. At the same time, the receptors that mediate phagocytosis of unopsonized Ft are unknown. This issue is of interest, as many studies of Ft-macrophage interactions have used the murine macrophage-like cell line J774A.1 (J774) [5 , 8 9 10 11 ]. In this system, multiplicities of infection (MOI) of 100:1 or more are required to achieve infection [5 , 12 13 14 ], which contrasts markedly with the ability of a few as 10 organisms to cause severe, fatal disease in mice and humans [1 , 15 ]. Recent data suggest that freshly isolated human peripheral blood monocytes are also difficult to infect with Ft [6 , 16 ]. The molecular basis for this low infection efficiency is unknown, and receptors that confer binding and phagocytosis of Ft have not been defined. Understanding how Ft gains entry into macrophages is important, as mechanism of entry can alter microbe fate [17 ].
In this study, we compared directly the ability of Ft subsp. holarctica live-vaccine strain (LVS) to infect J774 cells, human monocytes, and monocyte-derived macrophages (MDM). We now show that MDM are significantly more susceptible to infection with unopsonized Ft than blood monocytes or macrophage-like cell lines. Moreover, we demonstrate for the first time a specific role for the macrophage mannose receptor (MR) in phagocytosis of unopsonized Ft and define distinct roles for MR and CD11b/CD18 in uptake of opsonized bacteria.
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Cultivation of bacteria
Ft subsp. holarctica LVS was obtained from Dr. Michael Apicella (University of Iowa). Bacteria were inoculated onto sheep blood cysteine heart agar from frozen stocks and incubated for 48 h in a humidified incubator at 37°C. Bacterial colonies were harvested from plates, washed twice in PBS, and quantified by measuring the absorbance at 600 nm. H. pylori strain 11637 was cultivated on pH 6 sheep blood agar plates (37°C, 5% O2) under microaerophilic conditions as we described [18
]. Staphylococcus aureus strain ALC 1435 [19
] was grown overnight with shaking at 37°C in tryptic soy broth as described previously [20
].
Mononuclear cell isolation and culture
Heparinized venous blood was obtained from healthy adult volunteers using a protocol approved by the Institutional Review Board for Human Subjects at the University of Iowa, and all participants provided informed consent. Mononuclear cells were isolated by centrifugation on Ficoll-Hypaque, washed twice in RPMI, resuspended in RPMI + 20% autologous serum (AS) at a concentration of 2 x 106/ml, and differentiated into macrophages by incubation in Teflon jars for 5–7 days at 37°C [21
]. Where indicated, MDM were treated with 1000 IU/ml rhIL-4 on Day 5 and incubated an additional 48 h to induce alternative activation [22
]. Monocytes were obtained by plating freshly isolated peripheral blood mononuclear cells onto chamberslides (Nunc, Rochester, NY). After 2 h at 37°C, monocyte monolayers were washed twice to remove nonadherent lymphocytes. J774 cells (obtained from the American Type Culture Collection, Manassas, VA) and MR-positive J774-E clone cells [23
] (obtained from Philip D. Stahl, Washington University, St. Louis, MO) were maintained in DMEM containing 10% heat-inactivated (HI) FBS and 2 mM L-glutamine.
Phagocytosis
J774 cells, monocytes, or MDM were plated onto chamberslides (104 cells/well) in tissue-culture medium and allowed to adhere for 2 h at 37°C. Thereafter, MDM and monocytes were washed twice with PBS to remove nonadherent lymphocytes, and all samples were infected with LVS in fresh medium at a MOI of 20:1 or H. pylori at a MOI of 10:1. After 1 h at 37°C, media were removed, cells were washed twice with PBS to remove any uningested, noncell-associated bacteria, and samples were processed for microscopy as described below. For experiments assessing receptor recruitment to forming phagosomes, infections were synchronized as we described previously [24
]. Briefly, Ft were bound to adherent MDM or monocytes by a 4-min, 12°C spin at 600 g, and phagocytosis was initiated by rapid transfer to a 37°C incubator. After 5 min at 37°C, samples were fixed and processed as described below.
Receptor blockade
To block certain lectin receptors, MDM were incubated with 0.1–3.0 mg/ml mannan, 0.1–3.0 mg/ml laminarin, or 10 µg/ml anti-MR blocking antibody in RPMI containing 2.5% AS or 10% HI-FBS for 15 min at 37°C prior to addition of bacteria. To block complement receptors (CRs), phagocytes were incubated with anti-CD18, anti-CD11b, anti-CD11a, or anti-CD18 and anti-CD11b blocking antibodies at 5–25 µg/ml in RPMI containing 2.5% AS or 10% HI-FBS at 37°C for 5 min prior to infection.
Microscopy
Infected phagocytes were processed for microscopy using our established methods [24
] with minor modifications. Cells were fixed with 10% formalin, permeabilized using –20°C methanol-acetone (1:1), and then blocked overnight in PBS supplemented with 10% horse serum and 1 mg/ml bovine serum albumin (BSA). Fixed and permeabilized cells were double-stained to detect Ft and CD11b (M1/70.15.11.5.2), MR, or lamp-1, respectively. In all cases, secondary antibodies were conjugated to FITC or rhodamine. Coverslips were mounted to glass slides in gelvatol, and samples were examined using an LSM-510 confocal microscope or an Axioplan2 fluorescence microscope (both from Carl Zeiss, Inc., Thornwood, NY).
To quantify directly internalization of cell-associated Ft and H. pylori, the differential staining method of Heesemann and Laufs [25 ] was used as we described previously [26 ]. Briefly, mononuclear phagocytes infected with LVS or H. pylori for 60 min at 37°C were chilled on ice, washed with cold PBS, and then incubated on ice for 30–60 min in PBS containing anti-LVS or anti-H. pylori antibodies. After additional PBS washes, cells were fixed and permeabilized using methanol (10 min, 25°C) and then rehydrated in PBS supplemented with 5 mg/ml BSA. Extracellular bacteria were stained red by incubating cells in rhodamine-conjugated secondary F(ab')2 IgG (30 min, 25°C). Fixed and permeabilized cells were subjected to another round of staining with primary antibody followed by secondary antibody conjugated to FITC. All assays were performed in triplicate, and samples were examined using phase and fluorescence optics. In each case, the number of green (total cell-associated) and red (bound, extracellular) bacteria was counted, and the number of intracellular bacteria was determined by subtraction. By this assay, 85–95% of all cell-associated bacteria were phagocytosed, and engulfment was ablated (<2% ingested organisms) by the F-actin destabilizing agent latrunculin B or by infection of cells at 4°C (data not shown). Data are presented as phagocytic indices (intracellular bacteria per 100 cells).
Mannose binding lectin
Ft LVS and S. aureus were incubated at a concentration of 107/ml in RPMI containing 0, 2.5, or 10% AS for 30 min at 37°C. After two washes with PBS, samples were solubilized in sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) sample buffer. Ten million cell equivalents from each sample were resolved by 8% SDS-PAGE and transferred to polyvinylidene difluoride membranes. MBL and Ft LPS were detected by immunoblotting using secondary antibody conjugated to horseradish peroxidase [26
]. Bands were developed using enhanced chemiluminescence reagents (Pierce, Rockford, IL).
Statistics
Data were evaluated using one-way ANOVA or by the unpaired Student’s t-test (with the f-test to assess variance). In all cases, P < 0.05 was considered significant.
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Figure 1. Differential infection of mononuclear phagocytes by Ft LVS. LVS at a MOI of 20:1 (A) or H. pylori at a MOI of 10:1 (B) was added to J774 cells in DMEM + 10% HI-FBS or to monocytes and MDM in RPMI + 10% HI-FBS. Phagocytic indices were scored after 1 h at 37°C. Data are the average ± SEM of three independent experiments performed in triplicate. *, P< 0.05; **, P < 0.01, versus MDM.
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Figure 2. MRs mediate phagocytosis of unopsonized Ft. (A) MDM in RPMI + 10% HI-FBS were preincubated with the indicated concentrations of mannan (man), laminarin (lam), or 10 µg/ml anti-MR antibody prior to addition of Ft at a MOI of 20:1. After 1 h at 37°C, phagocytosis was quantified as in Figure 1
. Data indicate the mean ± SEM from at least three independent experiments for each condition and are normalized to the no-antibody control (9±2 Ft/100 MDM). *, P < 0.01, versus control. (B) Confocal sections show enrichment of MR on forming (5 min) LVS phagosomes (arrowheads). (C) J774 and J774-E cells in DMEM + 10% HI-FBS were infected with LVS (MOI 20:1), and phagocytosis was quantified after 1 h at 37°C. Data are the mean ± SD of triplicate samples from a representative experiment. *, P < 0.01. (D) MDM were left untreated or exposed to IL-4 for 48 h to induce alternative activation (Alt. activ.) prior to infection with LVS at a MOI of 20:1 for 60 min. Data are the mean ± SEM from three independent experiments performed in triplicate. *, P< 0.05. (E) MDM and alternatively activated MDM were incubated in RPMI containing 10% HI-FBS in the presence and absence of 0.1 mg/ml mannan prior to addition of Ft at a MOI of 20:1, and phagocytosis was quantified after 1 h at 37°C. Data are the mean ± SD of triplicate samples from one experiment representative of three independent determinations. *, P < 0.05, versus no mannan control.
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Macrophages treated with IL-4 undergo a phenotypic shift and achieve a state of alternative activation, which favors humoral immunity and tissue repair and is accompanied by changes in expression of many receptors including MR [34 , 35 ]. As alternatively activated macrophages up-regulate MR, we predicted that these cells may exhibit enhanced phagocytosis of Ft. To test this hypothesis, we treated MDM with rhIL-4 prior to Ft infection. As shown in Figure 2D , IL-4-treated MDM ingested twice as many bacteria as resting MDM. The extent to which enhanced uptake of Ft is mediated by MR alone or MR in combination with other receptors remains to be determined. Nevertheless, it is noteworthy that infection of alternatively activated MDM was impaired significantly in the presence of 0.1 mg/ml mannan and that resting and IL-4-treated MDM ingested similar numbers of Ft by mannan-insensitive mechanisms (Fig. 2E) . Collectively, these data suggest that alternatively activated macrophages are highly susceptible to Ft infection.
Serum complement enhances phagocytosis of Ft LVS by monocytes and MDM
As monocytes were refractory to infection with unopsonized Ft, we assessed the effect of fresh serum on phagocytosis. As shown in Figure 3B
, supplementation of RPMI with 2.5% fresh AS supported robust infection of monocytes, and the number of bacteria engulfed increased 35-fold relative to monocytes in standard media. Fresh AS had a significant (but less marked) effect on infection of MDM (Fig. 3A)
. In this regard, it is of interest that monocytes and MDM ingested similar numbers of opsonized bacteria after 60 min at 37°C (67–75 Ft/100 cells). Comparable data were obtained when Ft were opsonized at 37°C in 10% fresh AS and then washed prior to incubation with phagocytes in serum-free medium (data not shown). The ability of fresh AS to augment phagocytosis was ablated by heating serum to 56°C for 20 min (a treatment known to inactivate complement components). Indeed, data obtained using HI-AS and HI-FBS were indistinguishable.
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Figure 3. Serum complement is required for optimal phagocytosis of LVS. (A and B) Ft LVS was added at a MOI of 20:1 to MDM (A) or monocytes (Monos; B) in RPMI supplemented with 10% HI-FBS or 2.5% fresh AS as indicated. Phagocytosis was quantified after 1 h at 37°C. Data are the average ± SEM from three to six independent experiments performed in triplicate. **, P < 0.01. (C) MDM in RPMI containing 2.5% AS were left untreated (Control) or incubated with 25 µg/ml anti-CD18, anti-CD11b, anti-CD11a, or a mixture of anti-CD18 and anti-CD11b blocking antibody prior to addition of Ft. Phagocytosis was quantified after 1 h at 37°C. Data are the mean ± SEM from three independent experiments performed in triplicate and are normalized to the no-antibody control (42±7 Ft/100 MDM). *, P< 0.02; **, P < 0.01. (D) Effect of 25 µg/ml anti-CD18 and anti-CD11b antibody on Ft infection of freshly isolated blood monocytes in RPMI containing 2.5% AS. Data are the mean ± SEM from three independent experiments performed in triplicate and are normalized to the no-antibody control (44±4 Ft/100 monocytes). **, P< 0.01. (E and F) Human MDM (E) and monocytes (F) were infected with LVS in RPMI + 2.5% AS for 5 min. Confocal sections show enrichment of CD11b on forming phagosomes (arrowheads).
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70% inhibition, Fig. 3C
) and nearly ablated infection of monocytes (Fig. 3D)
. Conversely, blockade of lymphocyte function-associated antigen-1 (LFA-1) with mAb to CD11a did not disrupt Ft internalization (P=0.11, Fig. 3C
). Taken together, our data document for the first time an important role for serum complement and CD11b/CD18 in Ft infection of human monocytes and confirm the ability of fresh serum to enhance infection of MDM [36
].
CR3 does not mediate phagocytosis of unopsonized Ft
CR3 confers uptake of opsonized bacteria by engaging fixed C3bi. However, this receptor can also mediate nonopsonic uptake of certain microbes by binding directly to LPS or other surface factors [37
]. Therefore, we examined whether CR3 was involved in phagocytosis of unopsonized Ft. In marked contrast to opsonized bacteria, blockade of CD11b and/or CD18 did not prevent MDM phagocytosis of unopsonized Ft (Fig. 4A
, P
0.53), and CD11b was not detected on forming phagosomes (Fig. 4B)
. Blocking antibody specific for CD11a were also without effect (Fig. 4A)
. We conclude that CR3 is dispensable for phagocytosis of unopsonized Ft.
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Figure 4. CD11b/CD18 does not mediate phagocytosis of unopsonized Ft. (A) MDM in RPMI containing 10% HI-FBS were left untreated or preincubated with 25 µg/ml anti-CD18, anti-CD11b, anti-CD11a, or a mixture of anti-CD18 and anti-CD11b antibody prior to addition of Ft at a MOI of 20:1, and phagocytosis was quantified after 1 h at 37°C. In all cases, blocking antibodies were without effect (P>0.05). Data are the mean ± SD of triplicate samples from a representative experiment and are normalized to the no-antibody control (6±3 Ft/100 MDM). Similar data were obtained in two other independent experiments. (B) MDM were infected with LVS in RPMI + 10% HI-FBS for 5 min at 37°C. Confocal sections show that CD11b was not recruited to forming phagosomes containing unopsonized bacteria (arrowheads).
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Figure 5. Contribution of MR to uptake of opsonized Ft. (A) MDM were infected with LVS in RPMI + 2.5% AS for 5 min. Confocal sections show MR on forming LVS phagosomes (arrowheads). (B) MDM in RPMI containing 2.5% AS were left untreated (Control) or preincubated with the indicated concentrations of mannan, laminarin, or 10 µg/ml MR blocking antibody prior to addition of Ft at a MOI of 20:1. After 1 h at 37°C, phagocytosis was quantified. Data indicate the mean ± SEM of at least three experiments performed in triplicate for each condition and are normalized to the no-antibody control (102±32 Ft/100 MDM, n=6). *, P = 0.02, versus control.
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Six lines of evidence demonstrate that phagocytosis of unopsonized Ft is mediated in large part by the macrophage MR. First, MR accumulated on forming Ft phagosomes in MDM. Second, blockade of MR with soluble mannan or specific receptor-blocking antibody significantly impaired Ft binding and internalization, but inhibition of another lectin receptor (dectin-1) did not. Third, the extent of inhibition we obtained for Ft is comparable in magnitude with the effect of mannan on phagocytosis of other pathogens whose uptake is MR-dependent, including L. donovani and M. tuberculosis [21 , 41 ]. Fourth, MR-positive MDM ingested at least fivefold more bacteria than did MR-negative monocytes; and infection was enhanced further by alternative activation, a treatment known to increase MR expression [34 ]. Fifth, enhanced infection of J774-E cells demonstrated a role for MR in Ft infection of murine macrophages. Finally, differential infection of monocytes, MDM, and J774 cells was not a result of underlying differences in phagocytic capacity, as all three cell types ingested large numbers of H. pylori. Receptors that mediate lectinophagocytosis of H. pylori have not been defined; however, we show here that neither mannan nor laminarin affected uptake of this organism. Similarly, the Ft ligands that engage MR are unknown. One possible candidate is the mannose-rich surface exopolysaccharide capsule [42 , 43 ].
Although the results of this study define an important role for MR in Ft infection, the fact that neither mannan nor anti-MR antibodies ablated Ft entry into MDM suggests that additional, as-yet-unidentified receptors also contribute to Ft internalization. Of interest in this regard are members of the scavenger receptor family [44 45 46 ]. These receptors bind a variety of polyanions and mediate internalization of a wide variety of particles including Gram-positive and Gram-negative bacteria [45 ]. Unlike MR, members of the scavenger receptor family are present on J774 cells and monocytes as well as macrophages [46 , 47 ]. In future studies, it will be important to determine whether scavenger receptors allow monocytes and J774 cells to ingest unopsonized Ft, albeit with low efficiency. Also of interest are lectin receptors of the dendritic cell-specific intercellular adhesion molecule-3-grabbing nonintegrin (DC-SIGN) family [32 ]. Like MR, these receptors bind mannose-rich ligands on M. tuberculosis, Leishmania, and other pathogens [32 ]. Although DC-SIGN is not present on MDM or other resting macrophages [48 ], murine SIGN-related 1 is present on macrophages in the spleen, and liver/lymph node-specific-SIGN is expressed on a subset of liver and lymph node endothelial cells and macrophages [49 ]. Therefore, it is attractive to predict that a cohort of mannose-binding lectin receptors may play important roles in tularemia via their ability to support infection of a wide variety of cell and tissue types.
Alternative activation of macrophages is distinct from classical activation, which is driven primarily by interferon-
and enhances microbicidal function [34
, 35
]. Alternative activation is driven by IL-4 and IL-13 and results in a phenotype of enhanced endocytosis and antigen presentation, which are characterized by increased expression of MR and major histocompatibility complex Class II as well as production of predominantly anti-inflammatory cytokines [34
, 35
]. In this regard, it is of interest that alternatively activated macrophages up-regulate DC-SIGN and scavenger receptors in addition to MR, and we show here that IL-4-treated MDM exhibit enhanced phagocytosis of unopsonized Ft. In healthy adults, alternatively activated macrophages are found in the lung [48
], and it is generally believed that the distinct phenotype of alveolar macrophages limits potentially damaging inflammation in this locale [50
]. The results of this study suggest that alveolar macrophages may be particularly susceptible to Ft infection and if so, could account in part for the low infectious dose of this organism when acquired via the aerosol route. Indeed, published data suggest that murine alveolar macrophages engulf Ft LVS more avidly than other macrophage types and support bacterial replication [51
]. In contrast, classically activated MDM retard replication of Ft subsp. novicida by enhancing phagosome-lysosome fusion [7
].
Our studies of complement-opsonized Ft are notable for several reasons. First, we show that 2.5% fresh AS was sufficient to confer opsonophagocytosis. Second, CD11b/CD18 accumulated on forming phagosomes and mediated internalization of opsonized Ft, but LFA-1 did not. Third, the extent of inhibition we document here for opsonized Ft is concordant with the effects of CR3-blocking antibodies obtained in other systems [52 , 53 ]. Our data are also in good agreement with the results of a recent study [36 ], which found that 10% human AB serum enhances MDM infection with opsonized Ft in a CR3-dependent manner. Moreover, we extended the findings of Clemens et al. [36 ] to show for the first time that freshly isolated human blood monocytes and MDM ingest similar numbers of opsonized Ft and that CD11b accumulated on forming phagosomes only under conditions of opsonization. Moreover, our demonstration that monocyte infection was nearly ablated by CR3 blockade is consistent with the fact that these cells are refractory to infection with unopsonized Ft {(Fig. 1A) and ref. [16 ]}. Finally, we show that the lectin pathway of complement did not efficiently opsonize Ft.
Like Ft, L. donovani and virulent strains of M. tuberculosis use MR and CR3 to gain entry into macrophages [21
, 41
, 54
]. The ability of several intracellular pathogens to engage these two receptors is significant, as mechanism of entry is an important regulator of microbe fate [17
]. Of particular note here is the fact that infection via MR and CR3 is considered a relatively "safe" route of entry, which results in minimal activation of host microbicidal systems. The signaling cascades downstream of these two receptors are not coupled to production of proinflammatory cytokines and at least for CR3, do not trigger a strong respiratory burst [17
]. Indeed, MR ligation can actively suppress tumor necrosis factor 
(TNF-) [55
], and this may account for the fact that Ft LVS-infected MDM secrete less TNF- than infected monocytes [16
]. Finally, recent data indicate that engagement of MR by M. tuberculosis is required for subsequent inhibition of phagosome-lysosome fusion [33
]. Whether MR influences maturation of the Ft phagosome or alters the rate or extent of phagosome escape is unknown. However, the fact that opsonized and unopsonized Ft replicate in human and murine macrophages [5
6
7
], together with the results of this study, support the notion that MR and CR3 are safe portals of entry.
We and others [24 , 56 ] have shown previously that during CR3-mediated phagocytosis, C3bi-opsonized particles sink into the body of the macrophage without elaboration of large pseudopodia. In marked contrast, Clemens et al. [36 ] found that opsonized Ft are captured by large, asymmetric pseudopodia, which project from the macrophage surface to ensnare bacteria. Taken together, these data suggest that "looping phagocytosis" may not be mediated by signaling pathways downstream of CR3. We show here that opsonized Ft recruits MR and CR3 to forming phagosomes, and as such, our data suggest that signaling downstream of MR may influence phagosome morphology. In support of this notion, periodate oxidation of Ft LPS and capsule prevents looping entry [36 ], and under these conditions, forming phagosomes exhibit a typical CR3-like morphology, whereas uptake of opsonized Ft, which were killed by other methods, appears unchanged. This issue merits further study, and it is also possible that periodate-sensitive Ft virulence determinants modulate phagosome morphology by receptor-independent mechanisms.
In summary, we have shown that Ft LVS infects MDM more efficiently than peripheral blood monocytes or J774 cells in standard media and demonstrate that infection efficiency is mediated, at least in part, by the ability of unopsonized Ft to engage MR. At the same time, our findings support a model in which MR and CR3 play distinct roles during opsonophagocytosis. Additional studies are needed to define the full array of binding interactions that allow Ft to infect mononuclear phagocytes and to define how differential receptor expression impacts infection and microbe fate in vivo and in vitro.
Received March 24, 2006; accepted May 15, 2006.
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