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1.
Fig. 5.

Fig. 5. From: CD14 cooperates with complement receptor 3 to mediate MyD88-independent phagocytosis of Borrelia burgdorferi.

Inside-out signals are not required for the phagocytosis of B. burgdorferi. (A) RAW264.7 cells were incubated with Bb914 (m.o.i. = 25, green histogram) or MnCl2 (1 μM, red histogram), washed, and stained with the mAb CBRM1/5. The cells were fixed and analyzed by flow cytometry. (B) RAW264.7 cells were incubated with increasing concentrations of MnCl2 (0.5 μM, orange histogram; 1 μM, red histogram) or left unstimulated (black histogram), washed, and assessed for their phagocytic activity using Bb914 (m.o.i. = 25). (C) CHO-CR3 (Upper) and CHO-CR3/CD14 cells (Lower) were incubated with Bb914 (green histograms) or MnCl2 (red histograms), as before. The cells were stained with the mAb CBRM1/5, fixed, and analyzed by flow cytometry.

Kelly L. Hawley, et al. Proc Natl Acad Sci U S A. 2012 January 24;109(4):1228-1232.
2.
Fig. 4.

Fig. 4. From: CD14 cooperates with complement receptor 3 to mediate MyD88-independent phagocytosis of Borrelia burgdorferi.

CD14 cooperates with CR3 to promote phagocytosis of B. burgdorferi. (A) CHO-CR3 and CHO-CR3-CD14 cells were grown in eight-well chamber slides and incubated with Zymosan labeled with Alexa Fluor 488 (m.o.i. = 5) or Bb914 (m.o.i. = 25) for 4 h. The cells were washed, fixed, permeabilized, and stained with phalloidin Alexa Fluor 594, followed by analysis by Apotome fluorescence microscopy. (B) CHO-CR3/CD14 cells grown in chamber slides were incubated with Bb914 (m.o.i. = 25) for 4 h in the presence of M1/70 mAb or an isotype control. The cells were fixed, stained with phalloidin Alexa Fluor 594, and analyzed by confocal microscopy. (C) Flow cytometry of phagocytosis by CHO-CR3/CD14 (Lower) and RAW264.7 cells (Upper) in the presence of MyD88-BP (red histograms) or the control peptide (black histograms). The cells were preincubated with the peptides at 50 μM for 1 h, before adding Bb914 (m.o.i. = 25).

Kelly L. Hawley, et al. Proc Natl Acad Sci U S A. 2012 January 24;109(4):1228-1232.
3.
Fig. 1.

Fig. 1. From: CD14 cooperates with complement receptor 3 to mediate MyD88-independent phagocytosis of Borrelia burgdorferi.

CR3 mediates phagocytosis of B. burgdorferi. RAW264.7 cells (A) and BMMs (B) were incubated with Bb914 at an m.o.i. of 50 and 10, respectively, for 4 h in the absence (black histograms) or presence (green histograms) of increasing concentrations of anti-CD11b antibody; the gray histograms indicate a 4 °C control to determine background binding levels. The experiments shown are representative of at least three independent experiments. (C) Monocytes isolated from human peripheral blood were incubated with Bb914 (m.o.i. = 10) for 4 h in the presence of increasing concentrations of M1/70. (Upper) Representative flow cytometry histograms in the presence (red histogram) of 10 μg/mL of M1/70 or an isotype control (black histogram); the gray histogram indicates the 4 °C control. (Lower) The number of Bb914-containing monocytes assessed by epifluorescence microscopy. *Student's t test, P < 0.05.

Kelly L. Hawley, et al. Proc Natl Acad Sci U S A. 2012 January 24;109(4):1228-1232.
4.
Fig. 3.

Fig. 3. From: CD14 cooperates with complement receptor 3 to mediate MyD88-independent phagocytosis of Borrelia burgdorferi.

CR3 alone can mediate binding but not internalization of B. burgdorferi by CHO cells. (A) Microscopic analysis of binding of B. burgdorferi by CHO-CR4 and CHO-CR3 cells. A total of 5 × 104 CHO-CR4 and CHO-CR3 cells were grown for 16 h in eight-well chamber slides and incubated with Bb914 for 6 h at an m.o.i. of 100. The cells were washed, fixed, and stained with rhodamine phalloidin (red) and DAPI (blue). (B) Flow cytometry of Bb914 interaction with CR4- and CR3-expressing CHO cells after 6 h at an m.o.i. of 100. The graph is representative of three independent experiments. (C) CHO-CR3 cells grown in eight-well chamber slides were incubated with Bb914 at an m.o.i. of 100 in the presence of 10 μg/ml of anti-CD11b blocking antibody or an antibody control. The cells were fixed and stained with phalloidin (red) and DAPI (blue) and analyzed by microscopy. (D) CHO-CR3 cells were grown in eight-well chamber slides and incubated with Bb914 as before at an m.o.i. of 100 for 6 h. The unfixed cells were incubated with murine-infected serum (1/400) followed by anti-mouse IgG Alexa Fluor 568. (Lower) DIC images.

Kelly L. Hawley, et al. Proc Natl Acad Sci U S A. 2012 January 24;109(4):1228-1232.
5.
Fig. 2.

Fig. 2. From: CD14 cooperates with complement receptor 3 to mediate MyD88-independent phagocytosis of Borrelia burgdorferi.

CR3 mediates phagocytosis of B. burgdorferi by BMMs. (A) Phagocytosis of Bb914 by BMMs generated from CD11b-deficient mice (blue histogram) or B6 controls (black histogram). The 4 °C binding control is represented by the gray histograms. The cells were tested at an m.o.i. of 1 (Upper) and 10 (Lower). (B) Confocal micrograph of a BMM showing colocalization of GFP (B. burgdorferi Bb914, green) and CD11b (blue). The small micrographs represent internalized B. burgdorferi (arrows) that displayed presence or absence of colocalization with CD11b. (C) In vivo phagocytosis of B. burgdorferi by CD11b-deficient and wild-type B6 mice injected intraperitoneally with 1 × 107 spirochetes. A B6 mouse was also injected with non-GFP (WT) B. burgdorferi to control for migration of inflammatory cells into the peritoneum. The peritoneal lavages were analyzed after 6 h by flow cytometry. Shown are representative results for a total of three CD11b-deficient and three control B6 mice.

Kelly L. Hawley, et al. Proc Natl Acad Sci U S A. 2012 January 24;109(4):1228-1232.
6.
Fig. 6.

Fig. 6. From: CD14 cooperates with complement receptor 3 to mediate MyD88-independent phagocytosis of Borrelia burgdorferi.

Lyme carditis in CD11b-deficient mice involves increased inflammation and deficient control of B. burgdorferi. CD11b-deficient and wild-type controls were infected with 1 × 105 spirochetes by s.c. injection in the midline of the back. (A and B) After being euthanized, the mice were analyzed histologically for signs of arthritis (B) and carditis (A and B). *Student's t test, P < 0.05. n = 11 in each group. (C) Percentages of hearts positive for B. burgdorferi recA targets by qPCR. *χ2, P < 0.05. n = 16 in each group. (D) The levels of tnf gene expression in the hearts of the infected mice were analyzed by qRT-PCR relative to actin levels. *Student's t test, P < 0.05. n = 11 in each group. The results represent two to three independent experiments with 5–6 mice per group. (E) TNF production by B6 and CD11b-deficient BMMs in response to B. burgdorferi stimulation. BMMs were stimulated with live spirochetes at increasing m.o.i. for 16 h, followed by the analysis of the stimulation supernatants for TNF by ELISA.

Kelly L. Hawley, et al. Proc Natl Acad Sci U S A. 2012 January 24;109(4):1228-1232.

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