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. 2011 Aug;55(2):211-20.
doi: 10.1016/j.cyto.2011.04.009. Epub 2011 May 12.

Non-FcεR bearing mast cells secrete sufficient interleukin-4 to control Francisella tularensis replication within macrophages

Prea Thathiah  1 , Shilpa Sanapala, Annette R Rodriguez, Jieh-Juen Yu, Ashlesh K Murthy, M Neal Guentzel, Thomas G Forsthuber, James P Chambers, Bernard P Arulanandam
Affiliations

Non-FcεR bearing mast cells secrete sufficient interleukin-4 to control Francisella tularensis replication within macrophages

Prea Thathiah et al. Cytokine. 2011 Aug.

Abstract

Mast cells have classically been implicated in the triggering of allergic and anaphylactic reactions. However, recent findings have elucidated the ability of these cells to selectively release a variety of cytokines leading to bacterial clearance through neutrophil and dendritic cell mobilization, and suggest an important role in innate host defenses. Our laboratory has established a primary bone marrow derived mast cell-macrophage co-culture system and found that mast cells mediated a significant inhibition of Francisella tularensis live vaccine strain (LVS) uptake and replication within macrophages through contact and the secreted product interleukin-4 (IL-4). In this study, we utilized P815 mast cells and J774 macrophages to further investigate whether mast cell activation by non-FcεR driven signals could produce IL-4 and control intramacrophage LVS replication. P815 supernatants collected upon activation by the mast cell activating peptide MP7, as well as P815 cells co-cultured with J774 macrophages, exhibited marked inhibition of bacterial uptake and replication, which correlated with the production of IL-4. The inhibition noted in vitro was titratable and preserved at ratios relevant to cellular infiltration events following pulmonary challenge. Collectively, our data suggest that both primary mast cell and P815 mast cell (lacking FcεR) secreted IL-4 can control intramacrophage Francisella replication.

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Figures

Figure 1
Figure 1. Morphological examination and surface receptor expression of bone marrow derived mast cells and P815 mast cells
Mast cells were stained with Wright’s stain to demonstrate the presence of granules. Black arrows show presence of granules in BMMC (A), but not in P815 mast cells (B). 1 × 105 of each type of mast cell stained with fluorescent conjugated antibodies against FcεRI (PE) and cKit (FITC) and analyzed by flow cytometry (C–F).
Figure 2
Figure 2. Inhibition of F. tularensis LVS replication in mast cells and co-cultures
5 × 105 cells/well alone or in 1:1 co-cultures of macrophages: mast cells infected with LVS for 2 h, treated with gentamicin for an additional 2 h, cultured in fresh media or lysed, and dilution plated for bacterial enumeration. (A) LVS at 10 MOI. (B) LVS at 100 MOI. Results are representative of independent experiments repeated twice. *, P = 0.01 (two-way ANOA).
Figure 3
Figure 3. Infiltration of mast cells and macrophages into the lungs following intranasal LVS challenge
C57/BL6 mice were challenged intranasally with LVS (1600 CFU) and lungs collected at day 3 post-challenge, processed, and stained with fluorochrome conjugated antibodies to cKit, FcεRI, CD11b and F4/80. (A) Scatter plot analyses of total lung cells (mock or LVS infected) for mast cells and macrophages. Analysis of lung cells within the (B) cKit+ gate, (C) CD11bhi gate. (D) Percentages of infiltrating macrophages and mast cells in the lungs following LVS challenge. Percentages shown are for total lung cells. *, P < 0.05 (Student’s t-test).
Figure 4
Figure 4. Titration of inhibitory effect on LVS intramacrophage replication by P815 mast cells and bone marrow derived mast cells in co-culture with J774 macrophages
5 × 105 cells total/well infected (10 MOI) with LVS for 2 h, treated with gentamicin for an additional 2 h, cultured in fresh medium or lysed and dilution plated for bacterial enumeration. (A) J774 macrophages alone. (B) P815 mast cells or co-cultures of different ratios of macrophages:mast cells. (C) BMMC or co-cultures of different ratios of macrophages: mast cells. Results are representative of independent experiments repeated twice. (*, P = 0.01 (two-way ANOVA), P815 co-cultures and BMMC co-cultures compared with J774 macrophages alone at 24 h).
Figure 5
Figure 5. TNF-α production from macrophages alone and macrophages with decreasing ratios of P815 or bone marrow derived mast cells
TNF-α from 24 h cell culture supernatants from all conditions measured by ELISA. (A) J774 macrophages alone. (B) P815 mast cells alone and macrophages cultured with decreasing ratios of P815 mast cells. (C) BMMC alone and macrophages co-cultured with decreasing ratios of BMMC. Results are representative of independent experiments repeated twice. (*, P = 0.01 (two-way ANOVA), LVS infected P815 mast cells or co-cultures and BMMC or cocultures compared with mock infected cells at 24 h, *, P = 0.003 (two-way ANOVA), LVS infected P815 cells or BMMC compared with LVS infected J774 cells at 24 h).
Figure 6
Figure 6. IL-4 production from macrophages alone and macrophages with decreasing ratios of P815 or bone marrow derived mast cells
IL-4 from 24 h cell culture supernatants from all conditions measured by ELISA. (A) J774 macrophages alone. (B) P815 mast cells alone and macrophages co-cultured with decreasing ratios of P815 mast cells. (C) BMMC alone and macrophages co-cultured with decreasing ratios of BMMC. Results are representative of independent experiments repeated twice. (*, P = 0.01 (two-way ANOVA), LVS infected P815 mast cells or co-cultures and BMMC or co-cultures compared with mock infected cells at 24 h, *, P = 0.007 (two-way ANOVA), LVS infected P815 cells or BMMC compared with LVS infected J774 cells at 24 h).
Figure 7
Figure 7. Effect of IL-4/P815-spent supernatant on intramacrophage LVS replication
(A) Untreated, recombinant (r) IL-4 (5 or 25 ng/ml) treated, and co-culture with P815 mast cells. *, P=0.002 (two-way ANOVA). (B) Neutralizing anti-IL-4 antibody (50 μg/ml) treated J774 macrophages. *, P=0.004 (two-way ANOVA). All seeded at 5 × 105 cells/well. All conditions following infection with LVS (10 MOI) for 2 h, gentamicin treatment for an additional 2 h, then lysed or cultured in fresh media until the 24 h time point, lysed and dilution plated for bacterial enumeration. Results are representative of independent experiments repeated twice.
Figure 8
Figure 8. Inhibition of F. tularensis LVS replication in J774 macrophages by IL-4 released from activated P815 mast cells
5 × 105 J774 cells/well infected (10 MOI) with LVS for 2 h, treated with gentamicin for an additional 2 h, cultured in fresh medium or lysed and dilution plated. (A) LVS replication in J774 macrophages treated with filtered spent supernatants obtained from P815 mast cells treated with PBS, MP7 or MP17. *, P=0.01 (two-way ANOVA). (B) LVS replication in J774 macrophages treated with mock or LVS infected P815 filtered spent supernatants. *, P=0.006 (one-way ANOVA). (C) IL-4 detected in respective P815 cell spent supernatants as measured by ELISA. *, P=0.03 (two-way ANOVA). Results are representative of independent experiments repeated twice.
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