Limits...
Neutrophil recruitment to lymph nodes limits local humoral response to Staphylococcus aureus.

Kamenyeva O, Boularan C, Kabat J, Cheung GY, Cicala C, Yeh AJ, Chan JL, Periasamy S, Otto M, Kehrl JH - PLoS Pathog. (2015)

Bottom Line: They are rapidly mobilized to sites of infection where they help marshal host defenses and remove bacteria by phagocytosis.Within lymph nodes neutrophils infiltrate the medulla and interfollicular areas, but avoid crossing follicle borders.Our data reveal that neutrophils mobilized from the bone marrow in response to a local bacterial challenge dampen the early humoral response in the lymph node.

View Article: PubMed Central - PubMed

Affiliation: B-Cell Molecular Immunology Section, Laboratory of Immunoregulation, National Institute of Allergy and Infectious Diseases, National Institutes of Health, Bethesda, Maryland, United States of America.

ABSTRACT
Neutrophils form the first line of host defense against bacterial pathogens. They are rapidly mobilized to sites of infection where they help marshal host defenses and remove bacteria by phagocytosis. While splenic neutrophils promote marginal zone B cell antibody production in response to administered T cell independent antigens, whether neutrophils shape humoral immunity in other lymphoid organs is controversial. Here we investigate the neutrophil influx following the local injection of Staphylococcus aureus adjacent to the inguinal lymph node and determine neutrophil impact on the lymph node humoral response. Using intravital microscopy we show that local immunization or infection recruits neutrophils from the blood to lymph nodes in waves. The second wave occurs temporally with neutrophils mobilized from the bone marrow. Within lymph nodes neutrophils infiltrate the medulla and interfollicular areas, but avoid crossing follicle borders. In vivo neutrophils form transient and long-lived interactions with B cells and plasma cells, and their depletion augments production of antigen-specific IgG and IgM in the lymph node. In vitro activated neutrophils establish synapse- and nanotube-like interactions with B cells and reduce B cell IgM production in a TGF-β1 dependent manner. Our data reveal that neutrophils mobilized from the bone marrow in response to a local bacterial challenge dampen the early humoral response in the lymph node.

No MeSH data available.


Related in: MedlinePlus

Neutrophil depletion results in increased population of BLIMP1-YFP+ and GC B cells in immunized iLN.BLIMP1-YFP BM reconstituted mice were immunized with S. aureus subcutaneously, and injected with isotype control antibody or depleted of neutrophils using 1A8 antibody at days -1, 0 and 1 of immunization. ILNs were examined using epifluorescent microscopy, TP-LSM or flow cytometry. (A, B) Immunized mice were injected intravenously with Evans blue, and the iLNs of euthanized mice were exposed on a skin flip for imaging using stereomicroscope. Epifluorescent images of intact iLNs in immunized mice injected with (A) isotype control or (B) 1A8 antibody are shown. BLIMP1-YFP+ cells (green); blood vessels (red). Fluorescent images (top); corresponding bright field images (bottom left); scale bars, 400 μm. Lymph node borders and single blood vessels in IFZ are shown with dashed lines. Representative perivascular areas occupied with BLIMP1-YFP+ cells within the IFZ are outlined with white squares and their enlarged images are shown (bottom right); scale bars: 100 μm. (C) DsRed B cells or BM derived neutrophils were adoptively transferred at day 1–2 after immunization. TP-LSM images show LN follicles (red) from an immunized iLN of an isotype control-injected (left) or neutrophil-depleted (right) mouse. Scale bars, 20 μm. (D-F) Results from flow cytometry analysis of GC B cell (D) number and (E) percentage within the B220+ gate; and (F) BLIMP1-YFP+ GC B cell number within the B220+ gate. (G) Representative flow cytometry patterns of GC B cells from isotype control or 1A8 treated mice. (D-G) N = 4 iLNs; 3 independent experiments. Means ± SEM (H) BLIMP1-YFP B cells were isolated from the iLN and activated with LPS in vitro for 72 h. DsRed neutrophils were isolated from the BM and activated with LPS in vitro for 24 h. The cells were co-cultured for 2 h on ICAM-1/VCAM-1 coated surface. A confocal image of neutrophils (dsRed, red) interacting with a B cell (YFP, green) is shown. Scale bar: 5 μm. (I) DsRed BM derived neutrophils were adoptively transferred at day 7 after immunization, and recipient mice imaged 24 h later. A TP-LSM image of neutrophils (dsRed, red) interacting with B cells (YFP, green) is shown. Blood vessels (EB, gray). Scale bar: 10 μm. (J) Representative TP-LSM image of BLIMP1-YFP+ cells (green) localized in perivascular niches (S1 Movie) with neutrophils (red) migrating through the niches (left panel). Neutrophil tracks localized within the inside perimeter (outline, gray) of the niches (right panel). Scale bar: 20 μm. (H-J) Representative of 3 experiments.
© Copyright Policy
Related In: Results  -  Collection

License
getmorefigures.php?uid=PMC4401519&req=5

ppat.1004827.g006: Neutrophil depletion results in increased population of BLIMP1-YFP+ and GC B cells in immunized iLN.BLIMP1-YFP BM reconstituted mice were immunized with S. aureus subcutaneously, and injected with isotype control antibody or depleted of neutrophils using 1A8 antibody at days -1, 0 and 1 of immunization. ILNs were examined using epifluorescent microscopy, TP-LSM or flow cytometry. (A, B) Immunized mice were injected intravenously with Evans blue, and the iLNs of euthanized mice were exposed on a skin flip for imaging using stereomicroscope. Epifluorescent images of intact iLNs in immunized mice injected with (A) isotype control or (B) 1A8 antibody are shown. BLIMP1-YFP+ cells (green); blood vessels (red). Fluorescent images (top); corresponding bright field images (bottom left); scale bars, 400 μm. Lymph node borders and single blood vessels in IFZ are shown with dashed lines. Representative perivascular areas occupied with BLIMP1-YFP+ cells within the IFZ are outlined with white squares and their enlarged images are shown (bottom right); scale bars: 100 μm. (C) DsRed B cells or BM derived neutrophils were adoptively transferred at day 1–2 after immunization. TP-LSM images show LN follicles (red) from an immunized iLN of an isotype control-injected (left) or neutrophil-depleted (right) mouse. Scale bars, 20 μm. (D-F) Results from flow cytometry analysis of GC B cell (D) number and (E) percentage within the B220+ gate; and (F) BLIMP1-YFP+ GC B cell number within the B220+ gate. (G) Representative flow cytometry patterns of GC B cells from isotype control or 1A8 treated mice. (D-G) N = 4 iLNs; 3 independent experiments. Means ± SEM (H) BLIMP1-YFP B cells were isolated from the iLN and activated with LPS in vitro for 72 h. DsRed neutrophils were isolated from the BM and activated with LPS in vitro for 24 h. The cells were co-cultured for 2 h on ICAM-1/VCAM-1 coated surface. A confocal image of neutrophils (dsRed, red) interacting with a B cell (YFP, green) is shown. Scale bar: 5 μm. (I) DsRed BM derived neutrophils were adoptively transferred at day 7 after immunization, and recipient mice imaged 24 h later. A TP-LSM image of neutrophils (dsRed, red) interacting with B cells (YFP, green) is shown. Blood vessels (EB, gray). Scale bar: 10 μm. (J) Representative TP-LSM image of BLIMP1-YFP+ cells (green) localized in perivascular niches (S1 Movie) with neutrophils (red) migrating through the niches (left panel). Neutrophil tracks localized within the inside perimeter (outline, gray) of the niches (right panel). Scale bar: 20 μm. (H-J) Representative of 3 experiments.

Mentions: To analyze the impact of neutrophil influx on generation of early PC population in the LN we utilized mice expressing a BLIMP1-YFP transgene (MGI: 99655, S1 Table). BLIMP1-YFP mice were injected with isotype control or 1A8 antibodies and immunized with S. aureus bioparticles near the iLN. Consistent with previous characterization of PC development in BLIMP1-YFP mice [41], we identified YFPhi cells in the BM and the iLN of S. aureus immunized mice at day 7, but not at day 3 after immunization. As shown using epifluorescent stereomicroscopy at day 8 after immunization, YPFhi cells localized mostly in the perivascular regions in the MR and IFZ in the iLN of isotype control-injected mice (Fig 6A, white square). In the iLN of neutrophil-depleted mice, YPFhi cells were more abundant in the MR and IFZ, and more tightly packed around the blood vessels within these regions (Fig 6B, white square). Furthermore, YFPmed cells found in B cell follicles were more numerous in the neutrophil-depleted mice (Fig 6C). As shown by flow cytometry analysis the neutrophil-depleted mice had more B220+ cells (S6A Fig) and more GL7+Fas+ cells within B220+ gate per iLN than isotype control mice (Fig 6D and 6E). 2–6% of the B220+GL7+Fas+ cells were also BLIMP1-YFP+ (Fig 6F). These cells were enriched in the neutrophil-depleted mice and are likely the same cells observed in LN follicles using TP-LSM (Fig 6C). A typical flow cytometry pattern of the B220+ gated cells analyzed for GL7 and FAS expression from control mice and depleted mice is shown (Fig 6G).


Neutrophil recruitment to lymph nodes limits local humoral response to Staphylococcus aureus.

Kamenyeva O, Boularan C, Kabat J, Cheung GY, Cicala C, Yeh AJ, Chan JL, Periasamy S, Otto M, Kehrl JH - PLoS Pathog. (2015)

Neutrophil depletion results in increased population of BLIMP1-YFP+ and GC B cells in immunized iLN.BLIMP1-YFP BM reconstituted mice were immunized with S. aureus subcutaneously, and injected with isotype control antibody or depleted of neutrophils using 1A8 antibody at days -1, 0 and 1 of immunization. ILNs were examined using epifluorescent microscopy, TP-LSM or flow cytometry. (A, B) Immunized mice were injected intravenously with Evans blue, and the iLNs of euthanized mice were exposed on a skin flip for imaging using stereomicroscope. Epifluorescent images of intact iLNs in immunized mice injected with (A) isotype control or (B) 1A8 antibody are shown. BLIMP1-YFP+ cells (green); blood vessels (red). Fluorescent images (top); corresponding bright field images (bottom left); scale bars, 400 μm. Lymph node borders and single blood vessels in IFZ are shown with dashed lines. Representative perivascular areas occupied with BLIMP1-YFP+ cells within the IFZ are outlined with white squares and their enlarged images are shown (bottom right); scale bars: 100 μm. (C) DsRed B cells or BM derived neutrophils were adoptively transferred at day 1–2 after immunization. TP-LSM images show LN follicles (red) from an immunized iLN of an isotype control-injected (left) or neutrophil-depleted (right) mouse. Scale bars, 20 μm. (D-F) Results from flow cytometry analysis of GC B cell (D) number and (E) percentage within the B220+ gate; and (F) BLIMP1-YFP+ GC B cell number within the B220+ gate. (G) Representative flow cytometry patterns of GC B cells from isotype control or 1A8 treated mice. (D-G) N = 4 iLNs; 3 independent experiments. Means ± SEM (H) BLIMP1-YFP B cells were isolated from the iLN and activated with LPS in vitro for 72 h. DsRed neutrophils were isolated from the BM and activated with LPS in vitro for 24 h. The cells were co-cultured for 2 h on ICAM-1/VCAM-1 coated surface. A confocal image of neutrophils (dsRed, red) interacting with a B cell (YFP, green) is shown. Scale bar: 5 μm. (I) DsRed BM derived neutrophils were adoptively transferred at day 7 after immunization, and recipient mice imaged 24 h later. A TP-LSM image of neutrophils (dsRed, red) interacting with B cells (YFP, green) is shown. Blood vessels (EB, gray). Scale bar: 10 μm. (J) Representative TP-LSM image of BLIMP1-YFP+ cells (green) localized in perivascular niches (S1 Movie) with neutrophils (red) migrating through the niches (left panel). Neutrophil tracks localized within the inside perimeter (outline, gray) of the niches (right panel). Scale bar: 20 μm. (H-J) Representative of 3 experiments.
© Copyright Policy
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4401519&req=5

ppat.1004827.g006: Neutrophil depletion results in increased population of BLIMP1-YFP+ and GC B cells in immunized iLN.BLIMP1-YFP BM reconstituted mice were immunized with S. aureus subcutaneously, and injected with isotype control antibody or depleted of neutrophils using 1A8 antibody at days -1, 0 and 1 of immunization. ILNs were examined using epifluorescent microscopy, TP-LSM or flow cytometry. (A, B) Immunized mice were injected intravenously with Evans blue, and the iLNs of euthanized mice were exposed on a skin flip for imaging using stereomicroscope. Epifluorescent images of intact iLNs in immunized mice injected with (A) isotype control or (B) 1A8 antibody are shown. BLIMP1-YFP+ cells (green); blood vessels (red). Fluorescent images (top); corresponding bright field images (bottom left); scale bars, 400 μm. Lymph node borders and single blood vessels in IFZ are shown with dashed lines. Representative perivascular areas occupied with BLIMP1-YFP+ cells within the IFZ are outlined with white squares and their enlarged images are shown (bottom right); scale bars: 100 μm. (C) DsRed B cells or BM derived neutrophils were adoptively transferred at day 1–2 after immunization. TP-LSM images show LN follicles (red) from an immunized iLN of an isotype control-injected (left) or neutrophil-depleted (right) mouse. Scale bars, 20 μm. (D-F) Results from flow cytometry analysis of GC B cell (D) number and (E) percentage within the B220+ gate; and (F) BLIMP1-YFP+ GC B cell number within the B220+ gate. (G) Representative flow cytometry patterns of GC B cells from isotype control or 1A8 treated mice. (D-G) N = 4 iLNs; 3 independent experiments. Means ± SEM (H) BLIMP1-YFP B cells were isolated from the iLN and activated with LPS in vitro for 72 h. DsRed neutrophils were isolated from the BM and activated with LPS in vitro for 24 h. The cells were co-cultured for 2 h on ICAM-1/VCAM-1 coated surface. A confocal image of neutrophils (dsRed, red) interacting with a B cell (YFP, green) is shown. Scale bar: 5 μm. (I) DsRed BM derived neutrophils were adoptively transferred at day 7 after immunization, and recipient mice imaged 24 h later. A TP-LSM image of neutrophils (dsRed, red) interacting with B cells (YFP, green) is shown. Blood vessels (EB, gray). Scale bar: 10 μm. (J) Representative TP-LSM image of BLIMP1-YFP+ cells (green) localized in perivascular niches (S1 Movie) with neutrophils (red) migrating through the niches (left panel). Neutrophil tracks localized within the inside perimeter (outline, gray) of the niches (right panel). Scale bar: 20 μm. (H-J) Representative of 3 experiments.
Mentions: To analyze the impact of neutrophil influx on generation of early PC population in the LN we utilized mice expressing a BLIMP1-YFP transgene (MGI: 99655, S1 Table). BLIMP1-YFP mice were injected with isotype control or 1A8 antibodies and immunized with S. aureus bioparticles near the iLN. Consistent with previous characterization of PC development in BLIMP1-YFP mice [41], we identified YFPhi cells in the BM and the iLN of S. aureus immunized mice at day 7, but not at day 3 after immunization. As shown using epifluorescent stereomicroscopy at day 8 after immunization, YPFhi cells localized mostly in the perivascular regions in the MR and IFZ in the iLN of isotype control-injected mice (Fig 6A, white square). In the iLN of neutrophil-depleted mice, YPFhi cells were more abundant in the MR and IFZ, and more tightly packed around the blood vessels within these regions (Fig 6B, white square). Furthermore, YFPmed cells found in B cell follicles were more numerous in the neutrophil-depleted mice (Fig 6C). As shown by flow cytometry analysis the neutrophil-depleted mice had more B220+ cells (S6A Fig) and more GL7+Fas+ cells within B220+ gate per iLN than isotype control mice (Fig 6D and 6E). 2–6% of the B220+GL7+Fas+ cells were also BLIMP1-YFP+ (Fig 6F). These cells were enriched in the neutrophil-depleted mice and are likely the same cells observed in LN follicles using TP-LSM (Fig 6C). A typical flow cytometry pattern of the B220+ gated cells analyzed for GL7 and FAS expression from control mice and depleted mice is shown (Fig 6G).

Bottom Line: They are rapidly mobilized to sites of infection where they help marshal host defenses and remove bacteria by phagocytosis.Within lymph nodes neutrophils infiltrate the medulla and interfollicular areas, but avoid crossing follicle borders.Our data reveal that neutrophils mobilized from the bone marrow in response to a local bacterial challenge dampen the early humoral response in the lymph node.

View Article: PubMed Central - PubMed

Affiliation: B-Cell Molecular Immunology Section, Laboratory of Immunoregulation, National Institute of Allergy and Infectious Diseases, National Institutes of Health, Bethesda, Maryland, United States of America.

ABSTRACT
Neutrophils form the first line of host defense against bacterial pathogens. They are rapidly mobilized to sites of infection where they help marshal host defenses and remove bacteria by phagocytosis. While splenic neutrophils promote marginal zone B cell antibody production in response to administered T cell independent antigens, whether neutrophils shape humoral immunity in other lymphoid organs is controversial. Here we investigate the neutrophil influx following the local injection of Staphylococcus aureus adjacent to the inguinal lymph node and determine neutrophil impact on the lymph node humoral response. Using intravital microscopy we show that local immunization or infection recruits neutrophils from the blood to lymph nodes in waves. The second wave occurs temporally with neutrophils mobilized from the bone marrow. Within lymph nodes neutrophils infiltrate the medulla and interfollicular areas, but avoid crossing follicle borders. In vivo neutrophils form transient and long-lived interactions with B cells and plasma cells, and their depletion augments production of antigen-specific IgG and IgM in the lymph node. In vitro activated neutrophils establish synapse- and nanotube-like interactions with B cells and reduce B cell IgM production in a TGF-β1 dependent manner. Our data reveal that neutrophils mobilized from the bone marrow in response to a local bacterial challenge dampen the early humoral response in the lymph node.

No MeSH data available.


Related in: MedlinePlus