Participants, Materials, and Methods

Reagents The following reagents were obtained as noted: Hanks’ balanced salt solution (HBSS) with calcium and magnesium but without phenol red (Gibco), HBSS with 0.1% bovine serum albumin (BSA) (Sigma), gentamicin sulfate solution (10 mg/mL) (United States Biological), sterile MilliQue water (Millipore) filtered by use of a lipopolysaccharide (LPS)–free Acrodisc filter (Pall), LysoTracker red, CellTracker green, Syto16 green, Hoescht 33342, Prolong Gold with DAPI, and Alexa 488–BSA. A Via Gram staining kit (Molecular Probes) was also used

Antibodies The following antibodies were used in the present study: mouse anti–lysosomal-associated membrane protein (LAMP) 2 monoclonal antibody (MAb) (BD Pharmingen), rabbit anti-Salmonella immunogloubulin (Fitzgerald; lot P02050202), fluorescein isothiocyanate (FITC)–labeled rabbit anti–Salmonella flagella antigen (YVS 0703; Accurate Chemical and Scientific), rabbit anti-BSA IgG, Alexa 488– and Alexa 594–conjugated goat anti-mouse IgG, Alexa 488–conjugated goat anti-rabbit IgG (Molecular Probes), rabbit nonimmune rabbit IgG (Jackson-ImmunoResearch), and antigen affinity-purified rabbit anti-CR1 [9]

Salmonella species and microspheres S. montevideo (American Type Culture Collection) was grown overnight in Bacto nutrient broth (Difco) and quantified (0.5 OD₆₀₀ = 4.5×10⁸ cells/mL). Bacteria were gently pelleted, washed, and resuspended in HBSS. Wild-type (wt) S. typhimurium (SJW1103) and mutant strains either lacking flagella (SJW1368) or possessing flagella that could not rotate (SJW2960) were provided by May Macnab (Yale University) [10]. Sulfate-latex microspheres (1.0 μmol/L) (Interfacial Dynamics) were coated sequentially with Alexa 488–BSA (Molecular Probes), rabbit anti-BSA IgG, and, in some experiments, LPS (Escherichia coli; L3755; Sigma)

Human serum and cells Normal human serum (NHS) was obtained from healthy volunteers, and heat-inactivated serum (HIS) was prepared by heating NHS for 50 min at 56°C. Erythrocytes and PMNLs were isolated as described elsewhere [11] and were used within 30 min of purification. Cells and serum were obtained from autologous donors. The protocol for drawing blood from healthy volunteers was approved by the Beth Israel Deaconess Medical Center Institutional Review Board, and all participants provided informed consent

Assay for bacterial motility Bacteria (5×10⁷) were incubated with either buffer, 10% HIS in HBSS, or 10% NHS in HBSS for 10 min at 37°C. Time-lapse phase-contrast images recorded the “relative” distance traveled by individual organisms during 0.75 s. Images were analyzed by use of IPLab (version 3.9; Scanalytics)

Staining for bacterial flagella After exposure to either HIS or NHS, bacteria were treated with FITC-labeled rabbit anti-Salmonella flagella antigen (1:50) and 10 μg/mL Hoechst 33342 for 10 min and then were examined by fluorescence microscopy. In addition, bacteria opsonized under the standard conditions were stained as noted and analyzed by flow cytometry by use of a FACScan cytometer with CellQuest Pro software (version 4.01; BD Biosciences). Fluorescence-activated cell sorter buffer, unstained bacteria, and stained bacteria were examined by forward side scatter and fluorescence microscopy, for identification of the bacteria and determination of the gate for further analysis

Immune-adherence protocol S. montevideo bacteria were grown for 12 h at 37°C in nutrient broth, and bacteria were gently washed in HBSS. A total of 5×10⁷ S. montevideo bacteria were resuspended in 90 μL of HBSS, 10 μL of either NHS or HIS was added, and the suspension was incubated for 10 min at 37°C, for opsonization. Bacteria were washed once, 5 × 10⁶ erythrocytes were added to 1×10⁶ S. montevideo and the mixture was incubated in HBSS for 10 min at 37°C. The percentage of erythrocytes with immune-adherent S. montevideo was determined by light microscopy and ranged from 0% to 5% for HIS and from 70% to 75% for NHS

Phagocytic assay PMNLs (5×10⁵) were added to microfuge tubes containing 600 μL of HBSS/0.1% BSA. Serum-opsonized S. montevideo (1×10⁶), either free or immune adherent, were added to the PMNLs, and the mixture was incubated for 5 min at 37°C with end-over-end rotation at 8 rpm, as described elsewhere [12]. PMNLs were counted by light microscopy and analyzed for the percentage of phagocytosis (the percentage of PMNLs that had ingested intracellular S. montevideo) and the phagocytic index (the mean number of intracellular organisms in PMNLs that had ingested at least 1 organism). Buffer-treated organisms had a phagocytic index of 0%–11%, which was always less than that of HIS-opsonized organisms and was considered to be the baseline value

Intracellular killing assay Phagocytic mixtures were centrifuged to remove non–immune-adherent, noningested bacteria, and cells were subjected to hypotonic saline to lyse erythrocytes in the tubes that contained them. PMNLs were resuspended in gentamicin (10 μg/mL of HBSS) for 30 min at room temperature to kill extracellular, immune-adherent bacteria. Tubes were centrifuged at 3000 g for 3 min, and the PMNL pellets were suspended in 400 μL of HBSS. Samples were collected immediately (∼45 min after the initiation of phagocytosis) and again after an additional 60 min of incubation at 37°C. Viability sampling consisted of 4 replicate 7-μL aliquots, each diluted with 93 μL of sterile water and plated on separate petri dishes made with blood agar base number 2 (Difco). The plates for colony counts were incubated for 18 h at 37°C

Protocol for intracellular localization Purified PMNLs suspended in HBSS were loaded with 50 nmol/L LysoTracker for 30 min at 37°C, washed twice, and resuspended at a concentration of 1×10⁷ cells/mL in HBSS. S. Montevideo bacteria were loaded with 100 nmol/L Syto16 green for 30 min at 4°C in HBSS, washed twice with HBSS, opsonized with NHS, and resuspended at a concentration of 1×10⁸ bacteria/mL. PMNLs and opsonized bacteria with or without erythrocytes for immune adherence were mixed as described above. Aliquots (100 μL) from each sample were cytospun, and slides were mounted with Prolong Gold with DAPI. Cells were examined by use of an AX-70 Provis Olympus fluorescence microscope equipped with FITC and rhodamine filters (Olympus America) and UPLApo 40 × 1.0 Ph 3 and 100 × 1.35 Ph 3 objectives. Intracellular bacteria not associated with the lysosomal compartment fluoresced in the green channel alone, whereas intralysosomal bacteria fluoresced in both the green and the red channels. Images were acquired by use of a Retiga EXi digital camera (QImaging), and the images were further processed by use of IPLab. Alternatively, cells were fixed with 3.7% paraformaldehyde (EM Sciences) for 1 h, washed twice in HBSS, and permeabilized for 10 min in 0.2% saponin (Sigma-Aldrich). Cells were blocked with HBSS plus 4% NHS and 4% normal goat serum for 15 min at room temperature and were incubated with mouse anti–LAMP-2 MAb (5 μg/mL) for 20 min. Cells were washed twice with HBSS and incubated for 15 min with FITC-labeled rabbit anti–Salmonella flagella antigen (1:200). Cells were washed twice and stained with a mixture of Alexa 594–conjugated goat anti-mouse and Alexa 488–conjugated goat anti-rabbit IgG for 15 min. For colocalization studies, stacks of images (0.25 μm/step) were acquired by use of a Ludl z-axis motor, linear encoder, and filter wheels (LEP), which were controlled by software (IPLab). z-stacks were exported into Velocity (version 3.0; Improvision) for analysis

Statistical analysis Results are reported as means ± SD, and differences between samples were analyzed by use of Student’s t test. The level of statistical significance adopted was P<.05

Results

Effect of NHS on S. montevideo motility To potentially enhance immune adherence, we preopsonized the bacteria and washed them in buffer before adding erythrocytes. The removal of serum after opsonization minimizes the possibility that serum complement factor I, using CR1 as a cofactor, will degrade C3b on the bacteria and decrease immune adherence. This is not a consideration for immune-adherence clearance in vivo in humans, because clearance is very fast (t _1/2 = 5 min) [13], whereas, in vitro, the release of immune-adherent particles takes 8 times longer (50% release in 40 min) [14]

Unexpectedly, after opsonization with NHS, the bacteria seemed to be less motile. To confirm this finding, the protocol was repeated, and the movement of the bacteria was recorded by time-lapse light microscopy. Bacteria exposed to NHS were significantly less motile than those exposed to buffer or HIS (figure 1). Treatment with NHS did not affect Salmonella viability, as assessed by use of the Via Gram staining kit

NHS-induced shedding of bacterial flagella Flagellar function depends on an extracellular to intracellular proton gradient [15]. Under certain metabolic conditions or with excessive shear force, the flagella can be shed. To assess the possibility that NHS might induce the shedding of flagella on S. montevideo we used both flow cytometry and fluorescence microscopy. Bacteria that had been stained with a DNA dye were exposed to either buffer, HIS, or NHS and then were stained with FITC-labeled rabbit anti–Salmonella flagella antigen. NHS induced the shedding of flagella, with only green flagellin–positive stubs associated with the bacterial body, seen as a green outline in figure 2. The shed flagella accumulated in large clumps, suggesting that they had detached proximal to the bacterial surface. Samples that were analyzed by microscopy were also analyzed by flow cytometry, with similar results (figure 2). NHS opsonization rendered the S. montevideo less motile, and the bacteria became immune adherent, with consistently ∼70% of erythrocytes having ⩾1 bacillus attached (figure 3)

To assess whether NHS-induced loss of motility and becoming immune adherent to erythrocytes were functionally linked phenomena, we performed additional studies. S. typhimurium was used because of the availability of a wt strain (SJW1103) and 2 nonmotile mutant strains: 1 lacking flagella (SJW1368) and 1 possessing flagella that were nonfunctional because of a defective motor (SJW2960). With or without exposure to NHS, the wt S. typhimurium strain remained motile, whereas the 2 mutant S. typhimurium strains were nonmotile, as determined by microscopic observation. In a quantitative assay for immune adherence, each S. typhimurium strain was exposed to HIS or NHS and then was mixed with erythrocytes and examined for immune adherence by light microscopy. Sixty-one percent of flagella-lacking S. typhimurium and 81% of nonmotile S. typhimurium compared with only 33% of wt S. typhimurium became immune adherent. Although the wt S. typhimurium remained motile after opsonization, there was a marked enhancement of immune adherence in the nonmotile mutant strains. Thus, the NHS-induced flagellar shedding of S. montevideo may not have been essential for immune adherence, but, likely, it greatly enhanced it

Effect of immune adherence on phagocytosis of S. monte videoVarious species of bacteria are phagocytosed more efficiently when immune adherent [1, 16]. To determine whether immune adherence affected the uptake of S. montevideo by human PMNLs, we compared the ingestion of opsonized immune-adherent bacteria with that of opsonized non–immune-adherent S. montevideo. Immune-adherent S. montevideo were ingested more efficiently than were non–immune-adherent S. montevideo as measured by both the percentage of phagocytosis (50% vs. 29%) (figure 4A ) and the phagocytic index (3.7 vs. 2.7) (figure 4B )

Effect of of S. montevideoThe increased phagocytosis of immune-adherent S. montevideo might favor either the host or the pathogen. We therefore investigated the survival of S. montevideo within PMNLs after the bacteria in suspension were washed away and the immune-adherent, noningested bacteria were killed by gentamicin, manipulations that were completed 45 min after the initiation of phagocytosis. The viability of intracellular bacteria was assessed after 45 min and again after an additional 60 min of incubation (105 min total) at 37°C. These times were chosen because longer incubation times (120 min) resulted in all the bacteria being killed whether they had been free or immune adherent

PMNLs were lysed, and the lysate was diluted, plated in quadruplicate, and incubated for 18 h, to allow enumeration of viable colonies. The relative number of viable intracellular organisms at 45 min (figure 5) reflected the efficiency of phagocytosis for bacteria opsonized under different conditions (figure 4A and 4B ). During the subsequent 60 min of incubation, killing of previously immune-adherent S. montevideo was more efficient (60% decrease in colony-forming units between 45 and 105 min) (figure 5) than was killing of NHS-opsonized S. montevideo (27% decrease in colony-forming units during the same period) (figure 5)

Effect of immune adherence on the rate of fusion of bacteria-containing phagosomes with lysosomes Many intracellular bacterial pathogens, including S. typhi delay the fusion of the phagosome with the lysosomal compartment to subvert the host cell’s microbicidal mechanisms (reviewed in [17, 18]). We wondered whether the enhanced efficiency of killing of previously immune-adherent S. montevideo might relate to enhanced fusion of PMNL phagosomes with lysosomes, and we devised an assay to test this. Syto16 green was used to label S. montevideo whereas intracellular acidic compartments, predominately lysosomes, were labeled red with LysoTracker dye [19]. After incubation for 5 min at 37°C, to allow phagocytosis, and again after an additional 30 min of incubation (35 min total), samples were removed immediately. A representative postphagocytic PMNL is shown in figure 6A–6C to indicate how phagosomes were distinguished from phagolysosomes. The total number of intracellular bacteria was compared with those bacteria in the lysosomal compartment. Although the percentage of phagolysosomal fusion was higher at 5 min, if the bacterium had been previously immune adherent, by 35 min, the difference between free and immune-adherent bacteria was minimal (table 1)

Although LysoTracker is well accepted in the literature as a marker of lysosomes, there is the possibility that the dye marks other compartments with low pH. To address this concern, we repeated the experiment but replaced LysoTracker with anti–LAMP-2 MAb (figure 6E ) and replaced Syto16 green with rabbit anti-Salmonella antibody (figure 6D ). Because LAMP-2 has a diffuse distribution in PMNLs, the clustering around ingested salmonellae or the lack of clustering was relatively easy to identify and quantify (figure 6E ). Although immune-adherent bacteria were more rapidly ingested, there seemed to be a minimal effect on intracellular trafficking. To monitor more accurately the intracellular movement of phagosomes, we switched to Alexa 488–BSA–coated latex beads (microspheres) that had been opsonized with rabbit anti-BSA IgG and NHS. Being perfectly spherical and birefringent, microspheres were easy to identify by fluorescence and bright-field microscopy. To control for the presence of erythrocytes in the non–immune-adherent samples, we included an equivalent number of human erythrocytes that had been pretreated with antigen affinity-purified rabbit anti-CR1, which completely blocks immune adherence. Immune adherence stimulated the rate of phagocytosis of the microspheres (table 2). In striking contrast to the bacteria (whether free or immune adherent), at 35 min, the majority (83%) of the ingested microspheres remained in a LAMP-2–negative compartment, indicating that their phagosomes had not fused with lysosomes. These data suggest that, independent of being immune adherent and independent of antibody-complement opsonins, the molecular determinants of the target itself might determine intracellular trafficking within PMNLs

Effect of LPS, a Toll-like receptor (TLR) ligand, on phago somes containing latex microspheres To test whether the TLR ligand LPS could affect trafficking of phagosomes, PMNLs were incubated with opsonized beads with or without LPS for 5 min at 37°C and then were washed twice to remove the noningested microspheres. Cells were seeded on chamber slides and fixed every 10 min for the next 35 min. Although immobilized LPS had no significant effect on the percentage of phagocytosis or the phagocytic index (LPS beads, 19%; beads, 21%), there was a striking difference (figure 7A ) in the number of microspheres with fused phagolysosomes. We considered LAMP-2–positive microspheres to be only those microspheres that had a well-defined structured ring around them (figure 7D ), and not those microspheres that just displaced the LAMP-2–positive structures around them (figure 7A )

Effect of PMNLs on the trafficking specificity of various ingested particles We next asked whether phagocytosis of opsonized salmonellae would induce ingested microspheres to fuse with lysosomes. We mixed (1:1) opsonized salmonellae and opsonized Alexa 488 microspheres (lacking LPS) with PMNLs for 5 min of phagocytosis. After washing, PMNLs were seeded on glass for 15 min. Subsequently, the cells were fixed with paraformaldehyde, permeabilized, and stained for bacteria with anti-Salmonella MAb and anti–LAMP-2 MAb. A typical cell (figure 8) ingested both types of particles. Salmonellae and microspheres followed distinct pathways within the same cell: bacteria colocalized with LAMP-2, whereas microspheres remained in a LAMP-2–negative compartment

Discussion

We have investigated how immune adherence affects the killing of S. montevideo. Because erythrocyte-dependent immune adherence is unique to humans and a few other higher primates, our in vitro studies used human reagents. Although salmonellae can activate complement in the absence of antibody [20], it is likely that our sources of NHS contained specific antibody, because serum samples from some donors supported immune adherence better than serum samples from other donors

Nontyphoidal salmonellae have diverse virulence factors [21], and, thus, it was not surprising that exposure to NHS induced S. montevideo but not S. typhimurium to shed its flagella. Flagellar shedding not only promotes immune adherence, it also has the potential to disseminate an inflammatory response in vivo. Flagellin, the major flagellar protein (reviewed in [22]), binds TLR5 [23, 24] and, in terms of induction of proinflammatory genes, is the most important virulence factor of S. typhimurium [25]

Our data indicate that immune adherence accelerated uptake and intracellular trafficking of antibody complement–opsonized S. montevideo and microspheres (tables 1 and 2 and figures 4A 4B and 5). Nelson hypothesized that enhancement of immune adherence of phagocytosis might be due to immobilization of the bacteria [1], because trapping bacteria against surfaces had been shown to enhance the efficiency of phagocytosis [26] or because the erythrocyte stimulated the phagocyte. We observed minimal phagocytosis when HIS was used as an opsonin, and phagocytosis was not enhanced by the presence of bystander erythrocytes (figure 4A and 4B ). This finding argues against the idea that erythrocytes provide global stimulation of phagocytosis, but it does not rule out the possibility that direct stimulation occurs when the phagocyte contacts a particular erythrocyte to remove the immune-adherent target

Our experiments with antibody complement–opsonized microspheres confirmed that intracellular trafficking was not affected by immune adherence: ∼80% of ingested microspheres remained in LAMP-2–negative compartments regardless of whether they were free or immune adherent. However, the addition of a TLR ligand, LPS, to the target signaled for phagolysosomal fusion. It has been found by use of serum-opsonized green fluorescent protein E. coli as targets and mouse macrophages as effectors that an intact TLR signaling pathway leads to a higher rate of phagocytosis [27]. In contrast, we did not find that the addition of LPS to our microspheres enhanced either the percentage of phagocytosis or the phagocytic index. These discrepant results may relate to the fact that we used different targets and different effector cells. As was shown for murine macrophages [27], a single human PMNL could ingest a TLR-ligating target (bacteria) and a non–TLR-ligating particle (microsphere), and their trafficking was different: the bacteria stimulated rapid lysosomal fusion, and the microsphere did not. Thus, engagement of TLRs takes place in nascent phagosomes and signals for fusion of that particular phagosome with the lysosomal compartment

In summary, we have found that immune adherence facilitates phagocytosis of S. montevideo and that it likely is a host defense mechanism for other nontyphoidal Salmonella strains. During the course of these experiments, 2 novel findings were noted: first, opsonization with NHS caused S. montevideo but not S. typhimurium to shed its flagella, a finding that has implications for TLR5 signaling; and, second, the enhanced killing of immune-adherent S. montevideo was correlated with more-rapid phagolysosomal fusion in PMNLs. Intracellular trafficking, however, was not dictated either by host opsonins or by immune adherence, but rather by the biochemical composition of the target