 Hanks balanced salt solution (HBSS; Gibco). Antibiotics (all from Sigma) were added, when indicated, at the following concentrations: ampicillin, 100 μg/ml; kanamycin, 50 μg/ml; and streptomycin, 300 μg/ml.
Cell culture and macrophage/monocyte infections.Monocytes were isolated from citrated peripheral venous blood from healthy human volunteers by counterflow centrifugal elutriation, cultivated for HMDM in RPMI 1640 medium containing 10% heat-inactivated human AB serum (Sigma), 2 mM l-glutamine (Gibco), and macrophage colony-stimulating factor (10 ng/ml; a gift from Jay Stoudemire, Genetic Institute, Cambridge, Mass.), and incubated at 37°C in a humidified 5% CO2 atmosphere for 7 to 10 days as described elsewhere (7, 8, 9).
Monocytes incubated in the same medium for only 1 day were used as the source of 1-day-old monocytes. The murine macrophage-like cell line J774 was grown in RPMI 1640 medium supplemented with 10% fetal calf serum (Gibco), 2 mM l-glutamine, and penicillin-streptomycin (Gibco) in a humidified 5% CO2atmosphere at 37°C. Monocytes/macrophages were suspended in fresh medium in either 24-well culture plates, 6-well plates, or 100-mm-diameter tissue culture plates at a concentration of 106 cells/ml. The plates were washed to remove nonadherent cells before infection, and new medium without antibiotics was added. The cells were infected as described elsewhere (7, 8, 9). At selected intervals after infection, the medium was removed, and the macrophages/monocytes were washed and lysed with 0.1% Triton X-100. The numbers of viable bacteria were obtained by plating dilutions of the lysates on tryptic soy agar (TSA) plates. Colonies were counted after overnight incubation of the plates at 37°C.
Light microscopy analysis of infected macrophages and monocytes.Human or murine macrophages were seeded in tissue chamber slides (LabTek) and incubated at 37°C in a humidified 5% CO2 atmosphere. At selected intervals after infection, the slides were washed and stained using a LeukoStat stain kit (Fisher), which is a modification of the Wright's stain technique. The slides were examined under a light microscope.
Confocal microscopy.J774 cells were seeded on coverslips and infected as described above, using S. flexneri strains containing a plasmid expressing GFPuv (Clontech Laboratories). At selected intervals after infection, the medium was removed and the macrophages were washed, fixed, and stained for LAMP-1 (CD107a) as instructed by the manufacturer (Molecular Probes). Briefly, macrophages were fixed in buffered paraformaldehyde and permeabilized with 0.2% Triton X-100 in phosphate-buffered saline (PBS). The coverslips were then incubated at 4°C in blocking buffer (PBS containing 2% goat serum), washed in PBS, and incubated in 1:20 dilution of anti-LAMP-1 (mouse immunoglobulin G1 monoclonal antibody [MAb]). After being washed in PBS, the coverslips were counterstained with a 1:20 dilution of Alexa 594 goat anti-mouse immunoglobulin G (heavy plus light chain) conjugate antibody. After a final wash in PBS, the coverslips were mounted in medium containing 0.1 M n-propyl gallate (to prevent photobleaching) in glycerol (59% [vol/vol])–gelatin (0.9% [wt/vol]) and visualized by confocal microscopy using a Zeiss 410 instrument equipped with a krypton-argon mixed-gas laser. For green fluorescent protein (GFP) visualization, a 488-nm laser line was used for illumination and emission was detected with a 510- to 545-nm bandpass filter and a dichroic mirror to reflect wavelengths below 510 nm and above 550 nm; Alexa 594-tagged antibodies were irradiated with 568-nm laser, and emission was detected using a >610-nm-long bandpass filter and a dichroic mirror to reflect wavelengths below 600 nm. Phagosome lysosome fusion was considered to take place if colocalization of LAMP-1 and bacteria was observed.
Transmission electron microscopic (TEM) analysis of infection.At selected intervals following infection, monocyte/macrophage cell monolayers were washed with HBSS three times and prefixed with 4% paraformaldehyde–1% glutaraldehyde in 0.2 M sodium cacodylate buffer (SCB), pH 7.2, for 1 h at room temperature. The cells were then scraped off the tissue plate surfaces and placed in fresh prefixative and stored at 4°C for further processing. The samples were again washed three times with SCB and postfixed with 1% osmium tetroxide in SCB for 2 h as described elsewhere (8). The postfixed samples were further processed and embedded into Epon 812 (EPONATE 12; Ted Pella, Redding, Calif.). Ultrathin sections were made using a Leica Ultracut-S ultramicrotome. The sections were stained with uranyl acetate and lead citrate as described elsewhere (8) and were evaluated in a Philips 400 HM transmission electron microscope operating at an acceleration voltage of 80 kV.
LDH assays for measuring cytotoxicity.Monocytes/macrophages were seeded in 24-well plates and infected at a multiplicity of infection (MOI) of 30 bacteria/cell. Aliquots of the supernatants were collected and assayed for lactate dehydrogenase (LDH) release using a colorimetric Cytotox 96 kit (Promega Corp., Madison, Wis.) according to the manufacturer's instructions, with some modifications as described elsewhere (8, 9).
DNA fragmentation on agarose gels.Internucleosomal DNA fragmentation of infected monocytes/macrophages was measured as previously described (27, 28). The samples were electrophoresed on 1.2% agarose gels and stained with ethidium bromide as previously described (8, 9).
DNA analysis by flow cytometry.Macrophages were infected as described elsewhere (8, 9). After the last wash, nuclei from infected and uninfected macrophages were released from cells by treatment with 1% Triton X-100 in 0.1 M citric acid, stained with 10 μg of propidium iodide per ml, and analyzed on a FACScan flow cytometer (Becton Dickinson, Mountain View, Calif.) (8, 9).
Detection of levels of cytokines in the supernatant of infected macrophages.The release of cytokines in the culture supernatants of macrophages was measured at different times by using enzyme-linked immunosorbent assays (ELISA) for human or mouse IL-1β, tumor necrosis factor alpha (TNF-α), and human IL-10 and IL-12 as instructed by the manufacturer (Endogen). The reaction was stopped, and the intensity of the color was measured at 450 nm (correction wavelength was 570 nm) using a Titertek Multiscan ELISA reader as described elsewhere (7,8, 9).
Sereny reaction in guinea pigs.2457T-str containing a deletion in ipaH 7.8 (pWR700) was used to construct a second deletion in ipaH 4.5, generating the double-deletion mutant pWR730. Individual mutants ofipaH 4.5 (pWR710) andipaH 9.8 (pWR720) were also constructed in 2457T-str. The ipaH mutants were checked in invasion assays before administration into guinea pig eyes. Overnight plate growths of cells were harvested in PBS, 2.5 × 108 (low dose) to 109 (high dose) were inoculated into individual eyes of guinea pigs, and eyes were observed for development of keratoconjunctivitis as previously described (13). Diseased eyes were rated as follows: 0, no disease or mild irritation; 1, mild conjunctivitis; 2, keratoconjunctivitis without purulence; 3, fully developed keratoconjunctivitis with purulence; 4, eyes as in 3 but unusually swollen; 5, eyes as in 4 but with additional unusual inflammation around eyelids; 6, eyes as in 5 but with additional purulence and discharge. The unusual reaction seen with theipaH mutants made it necessary to extend the normal rating scheme for the Sereny reaction as described previously (13).
Statistical analysis.Statistical analysis was done by Student's t test using the INSTAT statistical analysis package (Graph Pad Software, Inc., San Diego, Calif.). Significance was a P value <0.05.
Behavior of IpaH deletions in the Sereny reaction.Deletions of ipaH 7.8 alone (pWR700) or in combination withipaH 4.5 (pWR730) (Table 1) in 2457T resulted in normal invasion of HeLa cells comparable to the wild-type strain. However, administration of pWR700 and pWR710 into guinea pig eyes resulted in a significantly exacerbated Sereny reaction; the eyes were considerably more swollen and redder, and they appeared highly irritated, with more inflammation than in the reaction seen with 2457T-str alone. Table 2 gives a representative example of one experiment with four guinea pigs (eight eyes) for each strain at each dose. Four such experiments were carried out with similar results. In the early stages of the disease, animals receiving the mutant strains often exhibited copious production of tears and had unusual redness and swelling around the eye and in the lower lid. The degree of inflammation, redness, and puffing of the eyes remained enhanced with the double mutant strain pWR730.
Evaluation of infection of ipaH mutant strains in HMDM and J774 cells.In J774