Monocyte Recruitment, Activation, and Function in the Gut-Associated Lymphoid Tissue during Oral Salmonella Infection

Mice

C57BL/6 mice were purchased from Charles River Laboratories. IFN-γ^−/−, IL-12p40^−/−, RAG^−/− mice and OT-II mice, all backcrossed more than nine generations on the C57BL/6 background, were bred at the Experimental Biomedicine Animal Facility of Göteborg University, Göteborg, Sweden. Mice were used at 8 to 12 wk of age and provided with food and water ad libitum. All experiments were performed using protocols approved by the Swedish government’s animal ethical committee and followed institutional animal use and care guidelines.

Bacterial strains

The S. enterica serovar Typhimurium χ3181, SR11 derivative χ4666 (40) was used for the studies shown in Fig. 1⇓, A and B, and Figs. 3⇓, 5⇓, and 8⇓. S. enterica serovar Typhimurium SL1344 and its enhanced GFP (eGFP)-expressing derivative SMO22 were used where bacterial uptake in vivo was examined (see Fig. 4⇓) (41). Strain 14028r (42) expressing OVA (43) was used for cytokine production and bacterial uptake experiments (Fig. 2⇓, C–E, and Fig. 6⇓B). Strain χ4550 expressing OVA-GFP or GFP (44) was used for in vitro infection in the Ag presentation assays (see Fig. 7⇓). Strain χ8554 (SR11 ΔasdA16 rpsL hisG) was used for in vivo infection of mice in Fig. 2⇓, C and D, and Figs. 6⇓, 7⇓, and 9⇓. Bacteria were grown in Miller’s Luria-Bertani (LB) broth or Lennox (χ8554) broth overnight at 37°C. For SL1344 and SMO22 the medium was supplemented with antibiotics (SL1344, 100 μg/ml streptomycin; SMO22, 50 μg/ml kanamycin and 100 μg/ml streptomycin). 14028r/OVA was grown on agar supplemented with 50 μg/ml carbenicillin. The bacterial suspension was diluted and the OD was measured at 600 nm. After centrifugation, the bacteria were resuspended at the appropriate concentration in sterile PBS. The actual bacterial dose administered was determined by the serial plating of bacteria on LB agar plates.

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FIGURE 1.

Populations differentially expressing Gr-1 and CD68 rapidly accumulate in the MLN and PP of orally infected mice. Cells from MLN (A–C and E) and PP (D) of naive mice and mice infected 5 days earlier were stained with 7-AAD, anti-CD68, anti-Gr-1, anti-CD11c, and one of the molecules shown in the histograms (E). Viable (7-AAD⁻) cells were then analyzed by five-color flow cytometry. A, Based on their expression of Gr-1 and CD68, cells were gated into CD68^intGr-1^high (R1) and CD68^highGr-1^int (R2) cells. B, Dendritic cells were defined as CD11c^highGr-1^low (R3) cells. C and D, cells from MLN (C) or PP (D) were gated as viable (7-AAD⁻) R1 and R2 cells as in A and the absolute number (MLN) and the percentage (PP) of the gated cells were examined on days (d) 3 and 5 postinfection. Bacterial recovery from the indicated organ on days 2, 3, and 5 postinfection is shown on the right. Each time point represents the mean value for a total of 6–7 mice examined in two independent experiments. Error bars indicate ± SD. E, Histograms show surface expression of the indicated molecules on gated R1, R2, and R3 cells from naive mice and from mice 5 days postinfection. The upper and lower numbers in the histograms are the mean fluorescence intensity of gated cells from infected and naive mice, respectively. The paucity of cells within the R1 gate in naive mice (A) made it impossible to accurately assess the level of expression of the molecules, and therefore naive levels were not determined in the R1 histograms. Dashed lines represent appropriate isotype control mAb. In A, B, and E similar results were obtained from PP (not shown). The data are representative of two or three independent experiments that examined a total of at least six mice per group.

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FIGURE 2.

Mono/mac are the main producers of TNF-α and iNOS during early infection with S. enterica serovar Typhimurium. A and B, cells from the MLN of naive mice or mice orally infected (Inf) 4 days earlier were stained with 7-AAD and anti-CD68, anti-Gr-1, anti-CD11c, anti-TNF-α, and anti-iNOS and analyzed by six-color flow cytometry. Intracellular TNF-α and iNOS staining of mono/mac (R2 in A), neutrophils (neutr) (R1 in B), and DC (R3 in B), defined as in Fig. 1⇑ were stained directly ex vivo at day 4 postinfection. The percentages of TNF-α, iNOS, and double positive cells are indicated in the quadrants. The right dot plot in A shows staining of mono/mac (R2) from naive mice. The staining of R1, R2, and R3 cells with isotype-matched control mAb for TNF-α and Ab for iNOS resulted in <1% and 0.1% positive cells, respectively, for all three populations (not shown). The number of neutrophils (R1) in naive mice was too low to quantify TNF-α and iNOS production. The results shown are representative of five independent experiments with a total of 10 mice. Complete data and statistical analysis are shown in Table I⇓. C–E, TNF-α (C) and IL-1β (D and E) production by monocytes, neutrophils, and DC sorted from infected mice and cultured ex vivo. Mice were orally infected with Salmonella and 4 days later cells from the spleen and blood (C and D) or MLN (E) of 10 mice were pooled and depleted of NK1.1⁺, CD19⁺, and TCR⁺ cells by MACS. Cells were stained for the surface expression of CD11b, CD11c, Ly6C, and Ly6G and sorted into Ly6C^highLy6G^low monocytes (purity >93%) and Ly6C^intLy6G^high neutrophils (purity >91%) as shown in Fig. 6⇓ and CD11c^high DC (purity >80%) as shown in Fig. 7⇓A (right dot plot). The sorted cells were incubated in medium alone (unstim, unstimulated; open bars) or pulsed with Salmonella for 2 h (restim, restimulated; black bars), washed extensively, and incubated in medium containing gentamicin. Supernatant was collected at 20 h for TNF-α and at 36 h for IL-β and was measured by ELISA. Results are expressed as mean ± SEM (pg/ml) of 1–3 independent experiments with duplicate samples. MACS-purified CD11b⁺ cells from naive mice are shown as a naive control. ND, not detected.

Infection of mice

Mice were given 0.1 ml of 1% NaHCO₃ intragastrically 10 min before infection. C57BL/6 or RAG^−/− mice were then infected intragastrically with 2–8 × 10⁷ bacteria in 200 μl of PBS. IFN-γ^−/− and IL-12^−/− mice are more susceptible to Salmonella and were given 5–8 × 10⁶ bacteria in 200 μl PBS intragastrically. Only IFN-γ^−/− and IL-12^−/− mice with a bacterial load similar to that of wild-type mice were included. In experiments where cell interactions with eGFP-expressing bacteria in vivo were studied (Fig. 4⇓), mice were infected intragastrically with 10⁹ SMO22 or SL1344 bacteria in 200 μl of PBS. In experiments using χ8554 (Fig. 2⇑, C–E, and Figs. 6⇓, 7⇓, and 9⇓), mice were infected intragastrically with 8 × 10⁸ bacteria in 200 μl of PBS. Mice were sacrificed after 2–5 days and the bacterial load in PP, MLN, spleen, and blood was determined by plating serial dilutions of single cell suspensions on LB or Lennox (χ8554) agar plates.

Cell preparation

Mice were sacrificed 2–5 days postinfection and the spleen, MLN, and PP were removed. Blood was collected from the heart by vascular perfusion with 5 ml of PBS containing 4 mM EDTA. The organs were digested with 0.45 mg/ml Liberase CI (Roche) in HBSS for 30 min at 37°C and pipetted into a single cell suspension or pressed through nylon mesh (TP filter). Splenocytes and blood were treated with 0.14 M NH₄Cl for 5 min at room temperature once or twice, respectively, to lyse RBC, and debris was removed by filtration. Cells were then washed three times with HBSS and resuspended in RPMI 1640 (Invitrogen Life Technologies) with heat-inactivated 10% FBS (PAA Laboratories). The total number of viable cells per organ was determined by trypan blue exclusion. For intracellular cytokine staining, 2 × 10⁶ cells/ml were resuspended in 5 ml of complete RPMI 1640 supplemented with 10% FBS and incubated at 37°C in the presence of 5 μg/ml brefeldin A (Sigma-Aldrich) for 4 h. For bacterial uptake studies in vivo, CD11b-expressing cells were enriched from PP and MLN pooled from 2–5 mice by magnetic separation using autoMACS (Milteny Biotech) after incubation with anti-CD11b beads (clone M1/70), (Milteny Biotech) according to manufacturer’s protocol.

Flow cytometry

Single cell suspensions were washed in FACS buffer (HBSS containing 3% FBS, 1 mM EDTA, and 10 mM HEPES) that was used throughout the surface staining procedure. Fc receptors on cells were blocked by incubating with the anti-FcγRII/III mAb 2.4G2 for 15 min at 4°C. Cells were then stained for 20 min at 4°C with the following mAbs that were biotinylated or conjugated to FITC, PE, allophycocyanin, allophycocyanin-Cy7, PE-Cy7, Pacific Blue, or Qdot605: anti-CD11c (clone HL3), anti-Gr-1 (clone RB6-8C5), anti-CD11b (clone M1/70), anti-F4/80 (clone CI:A3-1), anti-MHC-II (clone M5/114.15.2), anti-CD80 (clone 16-10A1), anti-CD86 (clone GL1), anti-CD62L (clone MEL-14), anti-NK1.1 (clone PK136), anti-CD19 (clone 1D3), anti-B220 (clone RA3-6B2), anti-TCR β-chain (H57-597), anti-CD4 (clone GK1.5), anti-Ly6C (clone AL-21), and anti-Ly6G (clone 1A8). The following anti-chemokine receptors were used: anti-CCR2 (clone MC21), anti-CCR6 (clone 1C12), anti-CXCR2 (clone 242216), and anti-CXCR3 (clone 220803). The Abs rat IgG1 (clone 8R3-34), IgG2a (clone R35-95), and IgG2b (clone A95-1) were used as isotype controls and biotinylated mouse anti-ratIgG2a as the secondary Ab. All mAb except anti-F4/80 (Caltag Laboratories), anti-CXCR2 and anti-CXCR3 (R&D Systems), and anti-CCR2 (generously provided by M. Mack, University of Regensburg, Regensburg, Germany), were purchased from BD Pharmingen. Anti-CD4 (clone GK1.5), purified in house, was conjugated to Qdot605 (Quantum Dot Corporation), and anti-Ly6G was conjugated to Pacific Blue (Molecular Probes) according to the manufacturer’s protocol. 7-Aminoactinomycin D (7-AAD; Sigma-Aldrich) was included in all stainings to define viable cells.

For staining with anti-CCR2 and anti-CCR6, cells were first blocked with 10% normal mouse serum (Sigma-Aldrich) for 15 min and stained with the primary Ab for 45 min. After washing, cells were incubated with a secondary biotin-conjugated Ab for 20 min, washed, and blocked with the FcγRII/III mAb 2.4G2 for 15 min. Finally, the cells were incubated with the additional mAbs to stain surface molecules and with streptavidin-allophycocyanin for 20 min.

When intracellular staining was performed, the samples were washed after surface staining and fixed in 2% formaldehyde in PBS for 20 min at room temperature. After the cells were washed in permeabilization buffer (HBSS containing 0.5% saponin and 0.5% BSA (Sigma-Aldrich)), which was subsequently used throughout the staining procedure, they were incubated for 30 min at room temperature with one or more of the following mAbs: anti-TNF-α (clone MP6-XT22), anti-IL12p40 (clone C15.6), anti-IFN-γ (clone 37895.11) (all purchased from BD PharMingen), anti-CD68 (clone FA/11; Serotec), S. enterica serovar Typhimurium O-4 (clone 1E6; Abcam), and the appropriate isotype control or iNOS Ab (M-19) (Santa Cruz Biotechnology). Staining with rabbit anti-iNOS was followed by incubation with allophycocyanin-conjugated anti-rabbit IgG (Santa Cruz Biotechnology) for 30 min at room temperature. An anti-Salmonella O-4 mAb was conjugated to Pacific Blue according to the manufacturer’s protocol.

Cells or bacteria (strains SMO22 and SL1344) were acquired on an LSR-II flow cytometer (BD Biosciences) using DIVA software (BD Biosciences) and analyzed using Flow Jo software (Tree Star). Whereas the absolute number of the cell populations is reported for the MLN and the spleen, the number and size of PP differs in individual mice and thus the percentage rather than the absolute number of cells in a given population was calculated.

Bacterial survival assay and Ag processing and presentation assays

Cell suspensions were made from the MLN, spleen, and blood pooled from 10 mice at day 4 postinfection as described above. Cells from the spleen and blood were subsequently pooled (to get enough cells to work with) and depleted of B, T, and NK cells by magnetic separation using autoMACS (Milteny Biotech) after incubation with a mixture of anti-CD19 beads, anti-CD90 (Thy1.2) beads, and anti-NK (DX5) beads according to the manufacturer’s protocol (Milteny Biotech). MLN cells were likewise depleted of T, B, and NK cells. After staining for flow cytometry, cells were sorted into CD11c^high DC, Ly6C^high monocytes, Ly6C^low monocytes, and Ly6G^high neutrophils using a FACSAria flow cytometer (BD Biosciences) and DIVA software (BD Biosciences). The different cell populations were seeded at 1.5 × 10⁵ cells/well in round-bottom 96-well plates in complete medium (RPMI 1640 Glutamax-1 containing 10% FBS (PAA Laboratories), 2 mM MEM sodium pyruvate, 20 mM HEPES, 0.05 mM 2-ME. and 2.5 μg/ml fungizone (all from Invitrogen Life Technologies)). For bacterial survival assays, sorted Ly6G^high neutrophils and Ly6C^high monocytes were infected with strain 14028r, centrifuged at 270 × g for 4 min, and incubated for 2 h at 37°C. The bacteria-to-cell ratio was between 5:1 and 18:1. After washing three times in complete medium containing 80 μg/ml gentamicin, the cells were resuspended in the medium and incubated at 37°C for an additional 1 or 18 h. Cells were lysed with 0.2% Triton X-100 with vigorous pipetting and incubated at room temperature for 10 min. The supernatant was then serially diluted and plated onto LB agar plates. Bacterial colonies were counted after overnight incubation at 37° C and the total number of bacteria recovered was calculated. As controls for determining the number of bacteria not internalized by the cells but viable in the supernatant, cells were preincubated with 20 μg/ml CCD (cytochalasin D) 1 h before the addition of bacteria and present for the initial 2 h of bacterial infection.

For Ag-presenting assays, titrated numbers of χ4550-OVA-GFP or χ4550-GFP (no OVA) were added to the cells. Alternatively, the OVA_323–339 peptide was added at a concentration of 0.1, 1, or 10 μg/ml. Cells were then centrifuged for 270 × g for 4 min and incubated at 37° C. After 2 h, the cells were washed three times in complete medium containing 80 μg/ml gentamicin and resuspended in 100 μl of medium with gentamycin. Finally, 2 × 10⁵ MACS-purified, CFSE-labeled OVA_323–339-specific CD4⁺ T cells from OT-II mice were added. The OT-II CD4⁺ T cells were negatively purified from pooled spleens plus MLN from naive mice using a CD4 isolation kit (Milteny Biotech) according to the manufacturer’s protocol and were labeled with CFSE. For CFSE labeling, cells were incubated with 5 μM CFSE (Molecular Probes) in PBS for 8 min at room temperature. After 3.5 days of coculture at 37°C, cells were harvested, stained with anti-CD4 and anti-TCRβ, and the percentage of divided CD4⁺ OT-II T cells was determined by flow cytometry.

Measurement of cytokines by ELISA

Sorted Ly6G^high neutrophils, Ly6C^high monocytes, and CD11c^high DC were resuspended in medium or infected for 2 h with χ4550-OVA or 14028r, washed, and treated as described for bacterial survival assays. After 20–36 h, as indicated in the appropriate figure legends, the plates were centrifuged and the supernatants were frozen at −20°C until assayed for cytokine content. The presence of TNF-α, IL-1β, and IL-12p70 in supernatants was determined by ELISA following the manufacturer’s protocol (BD Biosciences). To determine naive cytokine levels, CD11b-expressing cells were enriched from the spleen of naive mice by autoMACS after incubation with anti-CD11b beads (clone M1/70). The CD11b⁺ cells were seeded at 2 × 10⁵ cells/well in 96-well plates and incubated for 48 h before supernatants were frozen at −20°C.

Adoptive transfer and blocking experiments

Donor monocytes and neutrophils were negatively enriched using autoMACS and anti-CD49b (clone DX5) beads (Milteny Biotech) from pooled splenocytes and the blood of 10–15 RAG^−/− mice infected 3 days earlier with Salmonella χ4666. Donor T cells were negatively enriched from the pooled spleen and MLN of naive C57BL/6 mice using autoMACS and a CD4 isolation kit (Milteny Biotech). The donor monocytes, neutrophils, and T cells were then pooled and CFSE labeled as described above. Cells (10–15 × 10⁶) containing ∼35% T cells and ∼39% CD11b⁺NK1.1⁻ cells were injected i.v. into C57BL/6 mice that were infected 3 days earlier with χ4666. Some of the recipient mice were given 150 μg of the anti-MAdCAM-1 mAb MECA-367 (45) (BD Pharmingen) i.v. 6–8 h before cell transfer. Control animals received either 150 μg of the isotype control mAb 9B5 (anti-human CD44; kindly provided by A. Hänninen (University of Turku, Turku, Finland) or nothing. Twelve to 16 h after adoptive transfer, PP, MLN, spleen, and blood were removed from recipient mice and stained for flow cytometry as described above.

Phagocytic cells accumulate in the GALT after oral infection

The gut-draining lymphoid tissues PP and MLN are the first organs seeded by Salmonella after oral infection (27). However, little is known about the changes in the number, phenotype, and function of phagocytic cell populations, particularly the recently described monocyte subsets, responding to this oral pathogen in these organs. To address these issues, mice were orally infected with Salmonella and cells from PP and MLN were analyzed by five- to seven-color flow cytometry early during infection. Populations with differential expression of CD68 and Gr-1 responding to oral infection were apparent (Fig. 1⇑). In particular, two populations that were relatively rare in naive mice, CD68^intGr-1^high (where int stands for intermediate) and CD68^highGr-1^int cells (gate R1 and R2, respectively, in Fig. 1⇑), increased dramatically in the MLN and PP of infected mice starting at day 3 postinfection (Fig. 1⇑, A and C and D). Although the increase in both populations was large, CD68^highGr-1^int (R2) cells remained more abundant than gated R1 cells and comprised 1–3% of total cells per organ at day 5 postinfection.

To further characterize the two populations responding to oral Salmonella infection, the expression of a number of surface and costimulatory molecules was examined (Fig. 1⇑E). The gated CD68^intGr-1^high (R1) cells in infected mice lacked F4/80 and CD11c expression but had a very high level of CD11b. Few cells within this gate were positive for MHC-II, CD80, or CD86 at day 5 postinfection and the positive cells likely represent some spillover of R2 cells, as the junction between R1 and R2 gates in infected tissues becomes less absolute than in naive animals (Fig. 1⇑A). A fraction of R1 cells were positive for CD62L. Together, these data suggest that cells in the R1 gate are neutrophils (34, 38).

Although most cells within the R2 gate of infected MLN were CD11b^high, they had several features that distinguished them from R1 cells. This included the expression of F4/80 and a significant level of MHC-II by the majority of the gated cells. R2 cells from infected mice had increased CD80 and CD86 expression compared with naive mice, a fraction of the cells were CD62L⁺, and some expressed a low level of CD11c (Fig. 1⇑E). Thus, infection-induced R2 cells are phenotypically distinct from R1 cells.

Although the very low level of CD11c on gated R2 cells suggested that they were not conventional DC, which in the mouse are characterized by high surface expression of CD11c, MHC-II, and costimulatory molecules (46), the phenotype of gated R2 cells was compared with that of the CD11c^high population in the same infected individual (Fig. 1⇑, B and E). Consistent with previous reports, the CD11c^high cells expressed high levels of MHC-II, CD80, and CD86. Both myeloid and lymphoid DC are included in this gate as evidenced by the biphasic expression pattern for CD11b (Fig. 1⇑E), which corresponds to the CD8α⁺ and CD8α⁻ subsets (Ref. 46 and references therein). Moreover, additional studies showed that ∼20% of the CD11c^highCD11b^low DC express CD8α, which is consistent with previous data (46, 47, 48). Although most of the DC expressed CD68 (data not shown), their low Gr-1 expression and the nonoverlapping gating for Gr-1 on R2 vs R3 cells exclude DC from the R2 gate (Fig. 1⇑, A and B). Instead, most CD68^highGr-1^low cells in the PP and MLN of both naive and infected mice (Fig. 1⇑A, cells below the R2 gate) were DC expressing high levels of CD11c and MHC-II (data not shown). Furthermore, very few plasmacytoid DC identified as TCR⁻CD19⁻NK1.1⁻CD11b⁻CD11c^intB220⁺Ly6C⁺ (48, 49) were detected in naive MLN (∼0.06%), and they decreased to 0.01% of the total population in infected MLN. In addition, the CD11b⁻CD11c^int phenotype of plasmacytoid DC (48, 49) excludes them from the R1–R3 gates. Less than 10% of R1, R2, and R3 cells at day 5 postinfection were positive for the lymphoid markers CD19, TCRαβ, and NK1.1 (data not shown). B cells (CD19⁺) and T cells (TCR⁺ increased slightly in MLN and PP early during oral infection. In MLN, the absolute number of B and T cells increased 1.5-fold and 1.2-fold, respectively, at day 5 postinfection while the number of NK cells (NK1.1⁺TCRαβ⁻) remained constant (data not shown).

Together, the data in Fig. 1⇑ show that the predominant cellular change that occurs in the GALT after oral Salmonella infection is the rapid accumulation of cells with a phenotype distinct from neutrophils and DC. Moreover, the phenotype of R2 cells suggests they may be monocytes recruited from the blood, as has been shown in peritoneal or i.v. inflammation models and in lymph nodes draining inflamed skin (1, 2, 3, 4, 6, 7, 8, 37). Because monocyte refers to a population circulating in the blood and macrophage indicates a tissue resident differentiated cell, we refer to the cells that accumulate in infected PP and MLN as monocyte/macrophage (mono/mac).

The mono/mac that accumulate in PP and MLN are the main producers of TNF-α and iNOS during infection

To assess the function of the myeloid cells recruited to PP and MLN during oral Salmonella infection, their capacity to produce TNF-α and iNOS, effector molecules important in controlling Salmonella (30), were assessed by direct ex vivo double intracellular staining. Mono/mac producing TNF-α alone, iNOS alone, or both simultaneously were induced by Salmonella infection (Fig. 2⇑A and Table I⇓). Mono/mac were the predominant producers of these anti-bacterial molecules, accounting for 50–70% of all TNF-α⁺ and 80–90% of all iNOS⁺ cells in MLN. Moreover, 20–25% of the mono/mac that stain positive for iNOS were also positive for TNF-α.

In contrast to mono/mac, very few neutrophils or DC stained positive for iNOS (Fig. 2⇑B and Table I⇑). Similar results were also found in the PP and spleen of orally infected mice (Table I⇑). Some increase in cells staining positive for intracellular TNF-α was apparent among neutrophils and DC in infected mice (Table I⇑).

We also determined the ability of sorted monocytes, neutrophils, and DC from Salmonella-infected mice to secrete TNF-α, IL-1β, and IL-12p70 after ∼20 h (TNF-α) or ∼36 h of ex vivo culture. TNF-α and IL-1β were detected in the supernatant of cultured monocytes and neutrophils, whereas DC production of these cytokines was not apparent (Fig. 2⇑, C–E). Little if any increase in the amount of TNF-α was detected upon the restimulation of sorted monocytes with bacteria, and no increase was detected upon DC restimulation (data not shown). In contrast, the restimulation of monocytes or neutrophils with bacteria during the ex vivo culture resulted in enhanced IL-1β production (Fig. 2⇑, D and E). IL-12p70 was not detected in the supernatant of any of the sorted cell types cultured ex vivo with or without Salmonella (data not shown). Likewise, an increase in monocytes from the MLN or spleen of mice infected 3 or 5 days earlier with Salmonella that stained positive for intracellular IL-12p40 above the level detected in naive mice was not detected. Despite the fact that IL-12p70/IL-12p40 has a role during Salmonella infection (30), the cellular source(s) of this cytokine remain elusive.

Thus, mono/mac, defined as CD68^highGr-1^int cells, express MHC-II and costimulatory molecules (Fig. 1⇑E) and are the main sources of TNF-α and iNOS early during Salmonella infection. Moreover, the majority of mono/mac in infected tissues produce TNF-α or iNOS rather than both simultaneously.

Impaired expression of iNOS and MHC-II but not TNF-α in infected IFN-γ^−/− and IL12p40^−/− mice

We next asked whether iNOS and TNF-α produced by recruited mono/mac early during infection depended on IFN-γ and IL-12. First, the accumulation of mono/mac and neutrophils in the PP, MLN, and spleen of infected IFN-γ^−/− and IL-12p40^−/− mice with a bacterial burden similar to that of infected wild-type mice 4 days postinfection was not compromised (data not shown). Likewise, the frequency of mono/mac producing TNF-α in PP, MLN, and spleen was the same in wild-type, IFN-γ^−/−, and IL-12p40^−/− mice with a similar bacterial burden (Fig. 3⇓, A–C). In contrast, iNOS production by mono/mac in all three organs in infected IFN-γ^−/− and IL-12p40^−/− mice was nearly absent (Fig. 3⇓, A and B).

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FIGURE 3.

Defective iNOS and MHC-II but not TNF-α production in mono/mac from infected IFN-γ^−/− and IL12p40^−/− mice. Wild-type (WT), IFN-γ^−/−, and IL12p40^−/− mice were orally infected with Salmonella and 4 days (PP and MLN) or 5 days (spleen) later cells were stained with 7-AAD, anti-CD68, anti-Gr-1, anti-CD11c, and either anti-TNF-α and anti-iNOS (A and B) or anti-MHC-II (D) and analyzed by six- (A and B) or five-color (D) flow cytometry. Mono/mac and DC were gated as in Fig. 1⇑A. A, The dot plots show intracellular staining for TNF-α and iNOS on cells from the MLN of wild-type, IFN-γ^−/−, and IL12p40^−/− mice with a similar bacterial burden (shown in C). The numbers represent the percent positive cells in the quadrant. B, TNF-α (left) and iNOS (right) production by mono/mac in the PP, MLN, and spleen of infected wild-type, IFN-γ^−/−, and IL12p40^−/− mice with a similar bacterial burden (shown in C). TNF-α and iNOS production in naive mice is shown in Table I⇑. Error bars indicate ± SD. The p values (∗, p < 0.01; ∗∗, p < 0.001) are from the Mann-Whitney U test for infected C57BL/6 mice compared with infected IFN-γ^−/− or IL12p40^−/− mice. C, Bacterial recovery from PP, MLN, and spleen 4 days postinfection. D, MHC-II expression by mono/mac in PP and MLN (two left histograms) and DC in MLN (right histogram) in wild-type, IFN-γ^−/−, and IL12p40^−/− mice with a similar bacterial burden (shown in C) at day 4 postinfection. E, IFN-γ production by NK1.1⁺ cells (NK and NKT cells) and T cells (TCRαβ⁺NK1.1⁻) gated as in the upper dot plot in PP and MLN at day 4 postinfection (upper panel) is shown. Staining for IFN-γ in IFN-γ^−/− mice is used as a control (lower row). A–E Data were obtained in at least three independent experiments and each group contains 4–16 mice.

We also wished to determine whether the infection-induced increase in MHC-II on the mono/mac of infected wild-type mice was IFN-γ-dependent. Indeed, Salmonella-induced MHC-II up-regulation on mono/mac was severely compromised in IFN-γ^−/− and IL-12p40^−/− mice (Fig. 3⇑D). This is in contrast to MHC-II expression on DC, which was similar and high on CD11c^high (R3) cells in infected wild-type and knockout mice. Similar results were seen in the spleen (data not shown). We next examined the cellular source(s) of IFN-γ in the PP and MLN responsible for infection-induced mono/mac activation. These data revealed that NK and NKT cells (NK1.1⁺TCRα⁺) and some T cells (TCRαβ⁺NK1.1⁻) are the main producers of IFN-γ 4 days postinfection (Fig. 3⇑E). Thus, the induction of iNOS and MHC-II expression but not TNF-α production by mono/mac during oral Salmonella infection is dependent on IL-12 and IFN-γ, and NK/NKT cells and T cells are the predominant sources of IFN-γ in PP and MLN early during infection.

Uptake of eGFP-expressing Salmonella by phagocytic cells

To examine the relative uptake of bacteria by mono/mac and neutrophils in the same individual, mice were orally infected with Salmonella expressing eGFP and bacteria-associated cells were identified by flow cytometry. To get enough events to accurately assess the phenotype of eGFP⁺ cells, CD11b⁺ cells were first enriched by positive selection using MACS. In addition, an analysis of eGFP fluorescence negated using FITC-conjugated anti-CD68 (Fig. 1⇑A), so an alternate strategy using CD11b, Gr-1, and F4/80 was used to identify the phagocytic cells (Fig. 4⇓A) (7). To conclude that this strategy allowed identification of the neutrophil and mono/mac cells studied above, CD11b⁺Gr-1^highF4/80⁻ and CD11b⁺Gr-1^intF4/80⁺ were backgated into a CD68 vs Gr-1 plot (Fig. 4⇓B). This showed that the F1 and F2 populations defined by CD11b, Gr-1, and F4/80 identified the majority (63–95% in three independent experiments) of mono/mac (R1) and neutrophils (R2), respectively, with little cross-contamination between the two populations (Fig. 4⇓, A and B) (7).

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FIGURE 4.

Bacterial uptake in vivo by phagocytic cells in MLN and PP at day 4 postinfection. Mice were orally infected with Salmonella expressing eGFP or the parental strain not expressing eGFP and after 4 days CD11b⁺ cells were purified by MACS from MLN and PP. MACS-enriched cells were then stained with anti-CD11b, anti-Gr-1, and 7-AAD and anti-F4/80, anti-MHC-II, anti-TNF-α, anti-iNOS, or the appropriate control isotype and analyzed by five-color flow cytometry. A, CD11b-enriched cells were gated into CD11b⁺Gr-1⁺ cells and further gated into F4/80^high (F2) or F4/80^low (F1) populations. B, These two populations were then backgated into a CD68 vs Gr-1 plot to confirm that CD11b⁺Gr-1⁺F4/80^low cells correspond to CD68^intGr-1^high (R1 cells) and that CD11b⁺Gr-1⁺F4/80^high cells correspond to CD68^highGr-1^int (R2 cells). C, The percentage of eGFP⁺ cells within gated neutrophils (F1) and monocytes (F2) in PP (left) and MLN (right) of a Salmonella-infected mouse 4 days postinfection. The upper row is from a wild-type mouse infected with Salmonella expressing eGFP (Sal eGFP) while the middle row is a wild-type mouse infected with the parental Salmonella strain not expressing eGFP (Sal). The lower row of dot plots is from an IFN-γ^−/− mouse infected with Salmonella expressing eGFP. Data are representative of five independent experiments. The Mann-Whitney U test comparing C57BL/6 or IFN-γ^−/− mice infected with Salmonella expressing eGFP to mice infected with Salmonella not expressing eGFP was used for statistical analysis. The value of p ≤ 0.05 was obtained for all groups. Samples contained 34–2550 eGFP⁺ events. D, CD11b⁺Gr-1⁺ cells from the MLN and PP of mice infected 4 days earlier were gated as in the left dot plot in A. eGFP expression on gated iNOS⁺ (left) or TNF-α⁺ (right) cells is shown and the percentage of cells is indicated in each plot. E, The histograms show the expression of MHC-II on eGFP⁺ (open histogram) and eGFP⁻ (shaded histogram) CD11b⁺Gr-1^int cells (gated as in the dot plot) in PP of wild-type (WT) or IFN-γ^−/− mice infected 4 days earlier with eGFP⁺ Salmonella. The dotted line represents staining using the isotype-matched control mAb on eGFP⁻ cells (median value 97–162). The numbers show the mean fluorescence intensity for eGFP⁺ (top) and eGFP⁻ (bottom) CD11b⁺Gr-1^int cells in each plot. Cells were pooled from 2–4 mice. Data are representative of three independent experiments.

The results in Fig. 4⇑C show a slightly higher fraction of eGFP⁺ neutrophils (F1 cells) compared with mono/mac (F2) (∼1.6-fold difference) in the PP and MLN of infected wild-type mice. In infected IFN-γ^−/− mice, very similar fractions of neutrophils and mono/mac were eGFP⁺ in both PP and MLN (1.2-fold difference). The somewhat higher fraction of eGFP⁺ cells in the MLN of infected IFN-γ^−/− mice compared with wild-type mice reflects the higher number of bacteria recovered from IFN-γ^−/− mice in these experiments (data not shown) due to the compromised capacity to kill Salmonella in the absence of IFN-γ (30).

To investigate the accuracy of detecting cell-associated bacteria in wild-type and IFN-γ^−/− mice using eGFP fluorescence as the readout, two types of experiments were performed. First, the stability of eGFP in CD11b⁺ cells from infected wild-type and IFN-γ^−/− mice was assessed. These data showed similar eGFP stability, as 94 ± 6.3% (n = 7) and 94 ± 2.2% (n = 6) of bacterial colonies recovered from CD11b⁺ cells isolated from infected wild-type and IFN-γ^−/− mice, respectively, were eGFP⁺. Second, the detection bacteria by eGFP fluorescence was compared with the use of an anti-Salmonella O-4 LPS Ab. These studies revealed that 1.3–4.9% of CD11b⁺ cells from PP and MLN pooled from infected wild-type mice stained positive for O-4 reactivity, while eGFP fluorescence was detected in 0.4–2% of the cells in the same infected individuals. Identical percentages were obtained for infected IFN-γ^−/− mice. Although the down-regulation of eGFP fluorescence in vivo cannot be eliminated as a contributor to this difference, the greater reactivity of an anti-LPS Ab is not unexpected because it will recognize LPS on intact bacteria as well as bacterial degradation products and shed LPS. In contrast, the detection of bacteria using eGFP fluorescence relies on intact protein emitting fluorescence, a method that will not detect bacteria degraded to a point that negates eGFP fluorescence.

Very few eGFP⁺ events were found among TCRαβ⁺ or B220⁺ cells in infected tissues of the same animals, demonstrating that >80% of all bacteria-associated cells were either neutrophils or mono/mac (data not shown). The relationship between bacterial uptake and effector molecule production was also assessed directly ex vivo (Fig. 4⇑D). Approximately 30–40% of eGFP⁺ CD11b⁺Gr-1^int F2 cells were also iNOS⁺ (data not shown), demonstrating the tendency of bacteria-associated cells to produce this effector molecule. However, while ∼30–40% of all CD11b⁺Gr-1^int F2 cells expressed iNOS (Fig. 3⇑B), only a minor fraction of iNOS⁺ or TNF-α⁺ CD11b⁺Gr-1⁺ cells were eGFP⁺ (Fig. 4⇑D). This suggests that most iNOS and TNF-α-producing cells are bystander cells, as has been observed with other pathogens (5, 7, 12). In addition, bacterial uptake did not appear to influence the infection-induced up-regulation of MHC-II on mono/mac either in wild-type or IFN-γ^−/− hosts (see Fig. 4⇑E for PP; MLN data not shown). Similarly, bacterial uptake did not restore the ability of mono/mac from infected IFN-γ^−/− mice to produce iNOS (Fig. 3⇑A and data not shown).

Accumulation of cells in the blood of orally infected mice

Having established that neutrophils and cells with a phenotype suggesting a relationship to monocytes (3, 9, 13, 34, 50) (our so-called mono/mac population) accumulated and exerted effector functions in the gut-associated lymphoid tissues of orally infected mice, we asked when these cells appeared in the blood during infection and whether they were activated in the circulation. To this end, the populations defined in PP and MLN based on CD68 and Gr-1 expression were characterized in the blood of naive and Salmonella-infected mice. CD68 vs Gr-1 staining on blood cells revealed abundant R1 and R2 populations similar to those found in the tissues (Figs. 5⇓A and 1⇑A). In naive mice, R1 and R2 were 8 and 4%, respectively, of all leukocytes in blood. Both R1 and R2 populations accumulated in the blood as the infection progressed, increasing 2-fold and 4-fold, respectively, at day 3 postinfection and 2.5-fold and 4.5-fold, respectively, at day 5 (Fig. 5⇓, A and C).

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FIGURE 5.

Characterization of myeloid cells in the blood during Salmonella infection. Blood leukocytes from naive mice or from mice infected 4 days earlier were stained with 7-AAD, anti-CD68, anti-Gr-1, anti-CD11c, and one of the molecules shown in the histograms and analyzed by five-color flow cytometry. A, Using a gating strategy similar to that used for MLN (Fig. 1⇑A), neutrophils were identified as CD68^intGr-1^high cells (R1) and monocytes as CD68^highGr-1^int cells (R2). The gated R4 cells that are discussed in the text are also indicated. B, Dot plot of Gr-1 vs CD11c on cells from the blood of a naive and infected mouse showing the absence of CD11c^high cells (R3) and the presence of CD11c^int cells (R5). Data are representative of six naive and 10 infected mice examined in three independent experiments. C, Kinetics of R1, R2, and R4 cell accumulation in blood at days (d) 2, 3, and 5 postinfection. Data are the mean of 4–15 mice per time point from at least two independent experiments. Error bars are the SD. At these time points <100 to a maximum of 4000 bacteria were found in the blood collected from a mouse. D, Histograms show the expression of surface markers for the indicated cell populations in infected (open histogram) and naive (filled histogram) mice. Dashed histograms indicate staining with a isotype-matched control mAb. The numbers show the mean fluorescence intensity for infected (top) and naive (bottom) mice in each plot. All surface molecules were examined in at least two independent experiments on a total of 2–4 naive and 4–8 infected mice. E, TNF-α and iNOS production by R2 cells in the blood. The percentage of positive cells for either TNF-α or iNOS or for both is indicated. The data are representative of two independent experiments with a total of four naive and five infected mice. The isotype control for TNF-α showed <1% positive monocytes (not shown).

To further characterize the cell populations in blood during infection and compare them with the ones found in infected tissues, the expression of myeloid markers and costimulatory molecules on the blood populations was analyzed (Fig. 5⇑D). The phenotype of gated Gr-1^highCD68^int (R1) cells in the blood was very homogenous and similar in both infected and naive mice (Fig. 5⇑D). Essentially, all blood R1 cells expressed CD11b and CD62L but lacked F4/80, CD11c, MHC-II, CCR2, and the costimulatory molecules CD80 and CD86. In addition, gated R1 cells (as well as R2 and R4 cells) were negative for CD19, TCR, and NK1.1 (data not shown).

Like their tissue counterparts, blood CD68^highGr-1^int (R2) cells from infected mice were positive for CD11b, F4/80, and CCR2 (Fig. 5⇑D and data not shown). The adhesion molecule CD62L was expressed on some blood R2 cells in naive mice, and the fraction of CD62L⁺ cells increased in the blood of Salmonella-infected mice. Similar to R2 cells in the MLN of infected mice, R2 cells in the blood expressed much higher MHC-II in infected compared with naive animals. In contrast, however, the up-regulation of CD86 and particularly CD80 on R2 cells in the blood was modest compared with that seen in infected MLN (Figs. 1⇑E and 5⇑D).

Cells consistent with the phenotype of murine DC present in the tissues of naive and infected mice (CD11c^highMHC-II⁺ cells) were not found in the blood (Fig. 5⇑B and data not shown) (51). However, two populations of monocytes, Gr-1⁺CD62L⁺CCR2⁺ inflammatory and Gr-1⁻CD62L⁻CCR2⁻ resident monocytes, have been described in mouse blood (3). The Gr-1⁺CD62L⁺CCR2⁺ phenotype of gated R2 cells is consistent with the phenotype of inflammatory monocytes, while Gr-1⁻CD62L⁻ cells in R4 share features with resident monocytes (Fig. 5⇑, A and D) (3). R2 cells increased in the blood of infected mice while R4 cells did not (Fig. 5⇑C). Moreover, most of the cells within the R4 gate, putative resident monocytes, express an intermediate level of CD11c and up-regulate MHC-II during infection. This suggests a possible relationship to, or capacity to become, DC. Cells within gate R4 appear to be the best candidate for resident monocytes, as the CD11c^int cells in gate R5 in Fig. 5⇑B were mostly (>80%) NK1.1⁺CD11b^int NK cells (data not shown), consistent with other reports (51, 52). The remaining NK1.1⁻ cells in R5 were CD68^highGr-1^lowCD11b^int and fall within gate R4 in Fig. 5⇑A.

Given the efficient activation of mono/mac to produce large amounts of iNOS and TNF-α and to up-regulate MHC-II in the PP, MLN, and spleen during infection, we asked whether this activation already occurs in the blood of infected mice. As seen in Fig. 5⇑D, MHC-II was readily up-regulated on R2 cells, putative inflammatory monocytes in blood. However, no Salmonella-induced iNOS and little TNF-α above the level seen in naive mice were detected in this population (Fig. 5⇑E). Together, the data in Fig. 5⇑ show that two subsets of monocytes resembling resident (R4) and inflammatory monocytes (R2) can be identified in the blood of Salmonella-infected mice. R2 but not R4 monocytes increase in the blood during Salmonella infection and both have increased MHC-II expression but do not produce anti-bacterial effector molecules. Moreover, neutrophils increase in the blood of Salmonella-infected mice, while cells with the features of tissue DC are not readily apparent in the blood of naive or infected animals.

Bacterial uptake and Ag presentation capacity

To examine the phagocytic activity and killing capacity of monocytes and neutrophils, these cells were isolated from blood and spleen at day 4 postinfection, pooled, and depleted of T, B, and NK cells by MACS. The remaining cells were sorted into Ly6C^high monocytes (CD11b⁺Ly6G^lowLy6C^high) and neutrophils (CD11b⁺Ly6G^highLy6C^int) (Fig. 6⇓A). Because CD68 is expressed intracellularly and requires a staining procedure using fixation and permeabilization that kills the cells, we changed the gating strategy and used CD11b, Ly6C, and Ly6G to identify mono/mac and neutrophils (7, 8, 31, 34, 37, 53). Anti- TCR, CD19, NK1.1, and CD11c were also included in the staining to assure that no T cells, B cells, NK cells, or DC were included in the gates. To conclude that this strategy allowed identification of the mono/mac and neutrophils previously defined by CD68 and Gr-1, CD11b⁺Ly6G^lowLy6C^high monocytes and CD11b⁺Ly6G^highLy6C^int neutrophils were backgated into a CD68 vs Ly6G plot (Fig. 6⇓A). This showed that 79% of the CD11b⁺Ly6G^lowLy6C^high cells fell within the mono/mac (R2) gate and 97% of the CD11b⁺Ly6G^highLy6C^int cells fell into the neutrophil (R1) gate, with little cross-contamination between the two populations (Fig. 6⇓A).

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FIGURE 6.

Neutrophils and Ly6C^high monocytes phagocytose and kill Salmonella in vitro. Mice were infected with χ8554 and 4 days later cells from the spleen and blood of 10 mice were pooled and depleted of NK1.1⁺, CD19⁺, and TCR⁺ cells by MACS. Cells were stained for surface expression of CD11b, CD11c, Ly6C, and Ly6G and sorted into Ly6C^highLy6G^low monocytes and Ly6C^intLy6G^high neutrophils as shown in (A, left dot plot). Purity of the sorted cells was 95% for monocytes and 91% for neutrophils. A. The indicated Ly6C^highLy6G^low monocytes and Ly6C^intLy6G^high neutrophils (neut) were back-gated into a Ly6G vs CD68 plot to confirm that the monocyte and neutrophil populations identified by the Ly6C/G strategy correspond to those identified using Gr-1 (Ly6G) and CD68 (see Fig. 1⇑A). B, Sorted monocytes and neutrophils (Neu) were infected ex vivo with 14028r for 2 h in the absence or presence of CCD. The cells were washed and incubated in medium containing gentamicin for an additional 1 or 18 h. Cells were then lysed and the number of viable bacteria was determined by plating. Data are the mean of duplicate samples ± 1 SD. Similar results were obtained in two independent experiments.

The sorted monocytes and neutrophils were pulsed with Salmonella for 2 h, washed extensively, cultured for an additional 1 or 18 h in medium containing gentamicin, and the intracellular survival of bacteria was examined. After 3 h, both monocytes and neutrophils had phagocytosed bacteria but the neutrophils were more effective (Fig. 6⇑B), which is consistent with the in vivo data (Fig. 4⇑C). After 20 h, few viable bacteria were recovered from the two populations, showing that most of the phagocytosed bacteria had been killed. The use of CCD in parallel wells showed that the majority of bacteria were actively internalized rather than being attached to the cell surface (Fig. 6⇑B).

Having established that Ly6C^high monocytes were able to phagocytose bacteria in vitro, we wanted to examine whether they or Ly6C^low monocytes from infected mice were able to induce the proliferation of OT-II CD4⁺ T cells. To examine this, Ly6C^high monocytes, Ly6C^low monocytes, and DC from infected mice were sorted based on expression of CD11b, Ly6C, Ly6G, and CD11c (Fig. 7⇓A), pulsed ex vivo with Salmonella expressing OVA, and subsequently cocultured with CFSE-labeled OT-II CD4⁺ T cells. DC but not Ly6C^high or Ly6C^low monocytes induced proliferation of OT-II cells (Fig. 7⇓B). Bacterial titration showed that the T cell response peaked when DC were stimulated using a 5 to 1 bacteria to cell ratio, and that Ly6C^high monocytes were unable to induce a T cell response at all bacteria to cell ratios tested (Fig. 7⇓C). Furthermore, DC titration with a fixed bacteria to cell ratio (5 to 1) showed that proliferation of OT-II cells decreased as DC number declined (Fig. 7⇓D). The observed proliferation of DC was epitope-specific, because a lack of proliferation was observed when the cells were pulsed with Salmonella not expressing OVA (Fig. 7⇓D). Finally, pulsing with the OVA_323–339 peptide showed that Ly6C^high monocytes induced a modest proliferation of OT-II cells, while both Ly6C^low monocytes and DC efficiently induced proliferation (Fig. 7⇓B).

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FIGURE 7.

DC but not Ly6C^high monocytes (Mo hi) or Ly6C^low monocytes (Mo lo) process and present a Salmonella Ag on MHC class II. Cells from the blood and spleen of mice infected 4 days earlier with χ8554 were pooled, depleted of B, T, and NK cells, and stained with anti-CD11b, anti-CD11c, anti-Ly6C, and anti-Ly6G and a mixture of CD19, NK1.1, and TCRβ. A, Cells were sorted into Ly6C^high monocytes, Ly6C^low monocytes, and CD11c^high DC as shown. B, A total of 1.5 × 10⁵ cells/well were pulsed for 2 h with χ4550 expressing or not expressing OVA at a 5:1 bacteria to cell ratio. Alternatively, the cells were pulsed with 1 μg/ml OVA_323–339 peptide for 2 h. The cells were washed, resuspended in medium containing gentamicin, and 2 × 10⁵ MACS-purified, CFSE-labeled OT-II CD4⁺ T cells were added to the wells. After 3.5 days, the division of the CFSE-labeled T cells was assessed by flow cytometry. C, DC and Ly6C^high monocytes were pulsed at different bacteria to cell ratios with χ4550 expressing OVA for 2 h and processed as in B. D, DC infected at a 5:1 bacteria to cell ratio with χ4550 expressing OVA (circles) or not expressing OVA (square) for 2 h were washed and serially diluted 2-fold. OT-II cells were added and were processed as in B. Infection of the highest concentration of DC with χ4550 not expressing OVA induced <1.6% proliferation of the OT-II cells (square). Data are representative of 2–4 independent experiments. The purity of the sorted cells was 86% for DC, 93% for Ly6C^high monocytes, and 82% for Ly6C^low monocytes. Similar results were obtained with monocytes and DC purified from MLN (data not shown).

Together, these results show that Ly6C^high monocytes and neutrophils phagocytose and kill Salmonella in vitro. Moreover, and in contrast to DC, neither Ly6C^high or Ly6C^low monocytes induce OT-II T cell proliferation after pulsing with Salmonella expressing OVA although Ly6C^low monocytes induce OT-II proliferation after pulsing with OVA peptide.

Monocytes and neutrophils are recruited to PP despite blocking of MAdCAM-1

Monocytes and neutrophils in blood express the selectin CD62L (Fig. 5⇑D) and the α₄ integrin (37, 54, 55). CD62L and α₄ partnered with β₇, in particular, are ligands for MAdCAM-1. MAdCAM-1 is expressed on the high endothelial venules of PP and is critical for lymphocyte homing to this organ (45, 56, 57). To investigate whether MAdCAM-1 is also involved in monocyte and neutrophil recruitment to PP during Salmonella infection, blocking studies were performed. Although T cell recruitment to the PP of Salmonella-infected mice was abrogated in animals treated with the anti-MAdCAM-1 mAb MECA-367, monocytes and neutrophils accumulated in PP equally well in MECA-367-treated and untreated mice (Fig. 8⇓). MECA-367 blocked T cell recruitment to PP but not MLN (Fig. 8⇓. B and C) or spleen (data not shown) as predicted (45). Thus, unlike T cells, monocytes and neutrophils do not require the addressin MAdCAM to enter into PP in mice orally infected with Salmonella.

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FIGURE 8.

Recruitment of adoptively transferred Ly6C^high monocytes and neutrophils to PP during oral Salmonella infection occurs despite blocking MAdCAM-1. A total of 10–15 × 10⁶ CFSE-labeled monocytes, neutrophils, and T cells were injected i.v. into C57BL/6 mice orally infected 3 days earlier with χ4666. Half of the recipient Salmonella-infected mice were given anti-MAdCAM-1 mAb (MECA367) i.v. 6–8 h before cell transfer. Twelve to 16 h later, PP, MLN, spleen, and blood were removed and stained with anti-CD11b, anti-TCRβ, anti-CD19, anti-NK1.1, anti-Ly6C, and anti-Ly6G and analyzed by flow cytometry. A, TCRβ⁺CD11b⁻ cells were identified as T cells (left) and CD11b⁺TCRβ⁻CD19⁻NK1.1⁻ cells were further gated into monocytes (Ly6C^highLy6G^low) or neutrophils (Ly6C^intLy6G^high) (right) in PP as indicated. B and C, Recruitment of adoptively transferred T cells, monocytes, and neutrophils to PP on day 4 postinfection after blocking i.v with anti-MAdCAM-1 (MECA 367) (upper row) or not blocking (lower row) is shown. The percentage of CFSE⁺ cells within the population is indicated in the plots. Mice given an isotype control Ab (9B5; anti-human CD44) gave similar results as nontreated mice (not shown). Results are from five independent experiments with a total of 8–10 mice per group. Error bars are the SEM. The p value; (∗∗, p < 0.001) are from the Mann-Whitney U test for anti-MAdCAM-1-treated mice compared with unblocked or isotype-blocked mice.

Chemokine receptor expression on Ly6C^high monocytes, Ly6C^low monocytes, neutrophils and DC in blood, PP, and MLN of Salmonella-infected mice

To gain further insight into entry of myeloid cells into the gut lymphoid tissues during oral Salmonella infection, we next studied chemokine receptor expression because these receptor/ligand interactions provide important information that direct cell entry into lymphoid tissues (58). Thus, CXCR2, CXCR3, and CCR6 expression on myeloid cells from blood, PP and MLN were analyzed by flow cytometry at day 4 postinfection. Almost all blood neutrophils express CXCR2, while <20% of neutrophils in PP and MLN express this receptor (Fig. 9⇓). In contrast, few if any Ly6C^high monocytes in any of the immune compartments examined were CXCR2⁺. The fraction of cells positive for CXCR3 was relatively low for all cell types in all tissues examined, except for neutrophils in MLN where 13% were positive. Finally, CCR6 expression was only found on DC in PP and MLN. Thus, the high expression of CCR2 on Ly6C^high monocytes in blood (R2 cells; Fig. 5⇑D) is consistent with the role of this receptor and its ligand MCP-1 (CCL2) in the release of these inflammatory monocytes into the blood for subsequent entry into infected tissues, as described previously (3, 7, 12, 59). Moreover, the data suggest a potential role for CXCR2 in neutrophil migration and CCR6 in the migration of DC.

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FIGURE 9.

Chemokine receptor expression by neutrophils, Ly6C^high monocytes, Ly6C^low monocytes, and DC after oral Salmonella infection. C57BL/6 mice were orally infected with χ8554 and at 4 days postinfection CD19⁺ cells were MACS-depleted and the remaining cells were stained with 7-AAD and anti-CD11b, anti-Ly6G, anti-Ly6C, anti-CD11c, a pool of anti-NK1.1, anti-TCRαβ, and anti-CD19, and an Ab against a chemokine receptor (CXCR2, CXCR3, or CCR6). The expression of chemokine receptors on viable (7-AAD⁻) cells was assessed in the blood, PP, and MLN by seven-color flow cytometry. Histograms show surface expression of the indicated chemokine receptor on gated Ly6C^intLy6G^high neutrophils and Ly6C^highLy6G^low monocytes in all organs, on gated Ly6C^low monocytes in blood, and on DC in PP and MLN. Cells were gated as in Figs. 7⇑A and 8⇑A. Filled histograms represent staining with isotype-matched control Ab. Numbers are the mean value of chemokine receptor-positive cells with the isotype control subtracted (SEM) from 2–3 independent experiments using 10 infected mice/experiment that were divided into 1–3 groups.