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First published online October 30, 2006
doi: 10.1242/10.1242/jcs.03206

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Host cell Ca²⁺ and protein kinase C regulate innate recognition of Toxoplasma gondii

¹ Department of Pathobiology, School of Veterinary Medicine, University of Pennsylvania, Philadelphia, PA 19104, USA
² Department of Experimental Endocrinology, Medical University, Hanover, Germany
³ Department of Surgery, College of Physicians and Surgeons of Columbia University, New York, NY 10032, USA

View larger version (36K): [in a new window]   Fig. 1. MAPK signaling induced by T. gondii requires host Ca2+. (A) Fura-2-loaded macrophages were incubated in normal buffer (N) or BAPTA-AM (15 µM) in Ca2+-free EGTA medium (E/B) as described in Materials and Methods, then transferred to the Ca2+ imaging chamber, and stimulated with thapsigargin (thg; 1 µM, arrowhead) in normal buffer or Ca2+-free EGTA buffer, respectively, to release available intracellular Ca2+ stores. Images were captured at 5 frames per minute and 340/380 ratios were calculated and averaged to create the line tracings depicted. Averages are based on ratios collected from approximately 60 cells in each imaging field. (B-D) Macrophages were pre-treated with normal medium (N) or BAPTA-AM (15 µM) in Ca2+-free EGTA medium (E/B) for 45 minutes, washed, then treated with medium (m) as a control or infected with live T. gondii at a ratio of 5:1. Whole cell lysates were collected at the times indicated, and subjected to immunoblotting for phospho-ERK1/2 and phospho-p38 using individual antibodies specific to each MAPK (B, top panels). Membranes were then stripped and reprobed for total levels of ERK1/2 and p38 (bottom panels). Separate lysates were collected and used to immunoblot for phospho-MEK1/2 (C) or phospho-STAT3 (D). The membranes were stripped and reprobed for ß-actin to determine loading. Each experiment was repeated two to four times, and representative results are presented.

View larger version (50K): [in a new window]   Fig. 2. MAPK activation by parasite lysate, LPS, and CpG requires host cell Ca2+. Macrophages were depleted of Ca2+ by pre-treating with normal media (N) or BAPTA-AM (15 µM) in Ca2+ free EGTA medium (E/B) then treated with medium (m), STAg (A; 50 µg/ml), LPS (B; 100 ng/ml) or CpG (C; 1 µg/ml) for the times indicated. Whole cell lysates collected at the times indicated were used for immunoblotting with phospho-ERK1/2 and phospho-p38 antibodies. Blots were then stripped and reprobed for total MAPK. Results are representative of three experiments each.

View larger version (4K): [in a new window]   Fig. 3. Ca2+ is required for STAg-induced production of IL-12. Macrophages pre-treated with normal buffer (black bars) or BAPTA-AM (15 µM) in Ca2+ free/EGTA (white bars) were stimulated with STAg (50 µg/ml) for 5 hours in normal media or Ca2+ free EGTA medium, respectively. mRNA was isolated and reverse transcribed to cDNA, which was used in real-time PCR reactions for IL-12p35 and IL-12p40. All amplifications were conducted in triplicate and results are normalized to endogenous 18S and expressed as fold change in mRNA relative to untreated controls for each condition, according to the Ct method. Experiments were repeated at least three times with similar results. The data presented are from a representative experiment. *P0.01 by Student's t-test.

View larger version (65K): [in a new window]   Fig. 4. Ca2+ elevation enhances MAPK activation by parasite lysate or LPS. Macrophages were treated with medium (m) or stimulated with STAg (A; 50 µg/ml) or LPS (B; 100 ng/ml) either in the presence or absence of the Ca2+-mobilizing stimulus thapsigargin (1 µM) in normal Ca2+ media. Whole cell lysates were collected at the times indicated and immunoblotted for phospho-ERK1/2 and phospho-p38 then stripped and re-probed for total MAPK. Each experiment was repeated at least three times, and representative results are presented.

View larger version (12K): [in a new window]   Fig. 5. Microbial stimuli fail to induce an acute Ca2+ flux. Macrophages were loaded with Fura-2 and used for live cell Ca2+ imaging. (A, left panel) Fura-2-loaded macrophages were treated with thapsigargin (thg; 1 µM) after 3 minutes of baseline recording to demonstrate a typical Ca2+ response in these cells. Live T. gondii (A, right panel; 5:1), STAg (B; 50 µg/ml) or LPS (C; 100 ng/ml) was added to Fura-2-loaded macrophages following 3 minutes of baseline recording (arrows) and intracellular Ca2+ was recorded over the time period indicated. At the end of experiments in B and C, thapsigargin (thg; 1 µM) was added to demonstrate a positive response. Individual lines in each panel represent the intracellular Ca2+ level within each individual cell in the imaging field expressed as a 340/380 ratio. Experiments typically included 55-65 cells per imaging field. Recordings are representative of five to seven experiments for each condition.

View larger version (30K): [in a new window]   Fig. 6. T. gondii infection induces activation of conventional PKC. (A) Macrophages were infected with live T. gondii (5:1) or stimulated with media (m), STAg (50 µg/ml) or LPS (100 ng/ml) for the times indicated, then whole cell lysates were collected and fractionated by high-speed ultracentrifugation. Membrane fractions were used to immunoblot for PKC and PKCß as noted. Activation of PKC isoforms is determined by translocation to the membrane contained in the pellet fraction. (B) Densitometry of immunoblots in A, where data are represented as fold change over media-treated controls. Results are representative of seven experiments in which T. gondii induced a significant (P0.03) accumulation of both isoforms of PKC in the pellet fraction.

View larger version (43K): [in a new window]   Fig. 7. Conventional PKC regulate T. gondii-induced MAPK activation and production of IL-12. (A) PKCß–/– and WT macrophages were stimulated with medium (m) or STAg (50 µg/ml) for the times indicated and whole cell lysates were used for immunoblotting for phospho-ERK1/2 and phospho-p38. Blots were then stripped and reprobed for ß-actin (top panels) and PKCß (bottom panel). (B) WT macrophages were pre-treated with medium (m) or the conventional PKC inhibitor Gö6976 (1 µM), then treated with medium (m), infected with T. gondii (5:1) or stimulated with STAg (50 µg/ml). Whole cell lysates collected at the times indicated were immunoblotted for phospho-ERK1/2 and phospho-p38, then stripped and reprobed for ß-actin. (C) Macrophages treated with media (black bars), 5 µM (white bars), or 1 µM (grey bars) Gö6976 were infected (1:1) or stimulated with STAg (50 µg/ml) overnight, and the supernatants collected at 20 hours post-infection were assayed for IL-12p40 production by ELISA (error bars indicate s.e.m.). In each panel, results are representative of four to five experiments.