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Serum C3 Enhances Complement Receptor 3-Mediated Phagocytosis of Borrelia burgdorferi.

Hawley KL, Olson CM, Carreras-González A, Navasa N, Anguita J - Int. J. Biol. Sci. (2015)

View Article: PubMed Central - PubMed

Affiliation: 1. Department of Veterinary and Animal Sciences, University of Massachusetts Amherst, 01003 Amherst, MA, USA.

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Incubation of CHO-CR3, but not CHO-CR4, cells resulted in increased binding of the spirochete (Fig. 2A)... Furthermore, the presence of a blocking CD11b antibody (M1/70) also resulted in the reduction of B. burgdorferi phagocytosis in the presence of NMS in RAW cells (Fig. 2B)... Consistently, the presence of NMS during phagocytosis failed to increase the phagocytosis of B. burgdorferi in the absence of CD11b, compared to the use of HI serum (Fig. 2C), demonstrating that sera increased the phagocytosis of B. burgdorferi through its interaction with CR3... We argued that the CR3-dependent boost in phagocytosis in the presence of NMS would also reduce the production of TNF in response to B. burgdorferi... We stimulated BMMs with live B. burgdorferi at an m.o.i. of 25 for 16 h in the presence of NMS or C3-deficient serum... The presence of NMS significantly reduced the production of TNF by BMMs (Fig. 2D)... Importantly, the absence of C3 abrogated this inhibitory effect (Fig. 2D)... The effect of active serum on macrophages was specific for TNF, since the levels of IL-6 did not change in the presence of active serum (Fig. 2E)... Furthermore, the effect was specific of ligands that interact with CR3, since the presence of serum did not result in changes in the levels of TNF produced by BMMs in response to the TLR4 agonist, LPS (Fig. 2F)... Overall, these data demonstrate that CR3 is an anti-inflammatory phagocytic receptor both in the absence and the presence of the opsonin iC3b... In summary, our results show that serum C3-derived opsonins enhance the phagocytosis of B. burgdorferi mediated by CR3 further tempering the inflammatory output of macrophages induced by the integrin.

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Serum-mediated increase of B. Burgdorferi phagocytosis is CR3 dependent. (A) CHO-CR3 (bottom panel) and CHO-CR4 (top panel) cells were incubated with Bb914 (m.o.i. = 50) in the presence of HI (blue histogram) or NMS (red histogram). After 6 h, the binding of B. burgdorferi was determined by flow cytometry. (B) RAW cells were incubated with Bb914 in the presence of HI (blue histogram), NMS (red histogram) or NMS + 10 μg/mL of a blocking CD11b antibody (green histogram). After 2 h, the cells were analyzed by flow cytometry. (C) CD11b-deficient BMMs were incubated with Bb914 in the presence of HI (blue histogram) or NMS (red histogram). The cells were analyzed by flow cytometry after 4 h. The grey histograms represent a 4 ºC control. The data represent one of at least 3 independent experiments performed in triplicate. (D) BMMs from C57Bl/6 mice were stimulated with live B. Burgdorferi (m.o.i. = 25) in the presence of NMS (blue bar) or C3-deficient serum (red bar). TNF levels in the stimulation supernatants were measured 16 h later by ELISA. (E, F) BMMs were stimulated with B. burgdorferi (m.o.i. = 25) or LPS (100 ng/mL) in the presence of HI (green bar) or NMS (blue bar) for 16h. The supernatants were assessed for IL-6 (E) and TNF (F) by ELISA.
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Figure 2: Serum-mediated increase of B. Burgdorferi phagocytosis is CR3 dependent. (A) CHO-CR3 (bottom panel) and CHO-CR4 (top panel) cells were incubated with Bb914 (m.o.i. = 50) in the presence of HI (blue histogram) or NMS (red histogram). After 6 h, the binding of B. burgdorferi was determined by flow cytometry. (B) RAW cells were incubated with Bb914 in the presence of HI (blue histogram), NMS (red histogram) or NMS + 10 μg/mL of a blocking CD11b antibody (green histogram). After 2 h, the cells were analyzed by flow cytometry. (C) CD11b-deficient BMMs were incubated with Bb914 in the presence of HI (blue histogram) or NMS (red histogram). The cells were analyzed by flow cytometry after 4 h. The grey histograms represent a 4 ºC control. The data represent one of at least 3 independent experiments performed in triplicate. (D) BMMs from C57Bl/6 mice were stimulated with live B. Burgdorferi (m.o.i. = 25) in the presence of NMS (blue bar) or C3-deficient serum (red bar). TNF levels in the stimulation supernatants were measured 16 h later by ELISA. (E, F) BMMs were stimulated with B. burgdorferi (m.o.i. = 25) or LPS (100 ng/mL) in the presence of HI (green bar) or NMS (blue bar) for 16h. The supernatants were assessed for IL-6 (E) and TNF (F) by ELISA.

Mentions: To demonstrate that the enhanced phagocytosis of B. burgdorferi in the presence of NMS was specific to CR3, we used CHO cells transfected with human CR3 (CD11b/CD18; CHO-CR3) or CR4 (CD11c/CD18; CHO-CR4) as controls 1, 7. Incubation of CHO-CR3, but not CHO-CR4, cells resulted in increased binding of the spirochete (Fig. 2A). Furthermore, the presence of a blocking CD11b antibody (M1/70) 1 also resulted in the reduction of B. burgdorferi phagocytosis in the presence of NMS in RAW cells (Fig. 2B). We also used BMMs generated from CD11b KO mice. Consistently, the presence of NMS during phagocytosis failed to increase the phagocytosis of B. burgdorferi in the absence of CD11b, compared to the use of HI serum (Fig. 2C), demonstrating that sera increased the phagocytosis of B. burgdorferi through its interaction with CR3.


Serum C3 Enhances Complement Receptor 3-Mediated Phagocytosis of Borrelia burgdorferi.

Hawley KL, Olson CM, Carreras-González A, Navasa N, Anguita J - Int. J. Biol. Sci. (2015)

Serum-mediated increase of B. Burgdorferi phagocytosis is CR3 dependent. (A) CHO-CR3 (bottom panel) and CHO-CR4 (top panel) cells were incubated with Bb914 (m.o.i. = 50) in the presence of HI (blue histogram) or NMS (red histogram). After 6 h, the binding of B. burgdorferi was determined by flow cytometry. (B) RAW cells were incubated with Bb914 in the presence of HI (blue histogram), NMS (red histogram) or NMS + 10 μg/mL of a blocking CD11b antibody (green histogram). After 2 h, the cells were analyzed by flow cytometry. (C) CD11b-deficient BMMs were incubated with Bb914 in the presence of HI (blue histogram) or NMS (red histogram). The cells were analyzed by flow cytometry after 4 h. The grey histograms represent a 4 ºC control. The data represent one of at least 3 independent experiments performed in triplicate. (D) BMMs from C57Bl/6 mice were stimulated with live B. Burgdorferi (m.o.i. = 25) in the presence of NMS (blue bar) or C3-deficient serum (red bar). TNF levels in the stimulation supernatants were measured 16 h later by ELISA. (E, F) BMMs were stimulated with B. burgdorferi (m.o.i. = 25) or LPS (100 ng/mL) in the presence of HI (green bar) or NMS (blue bar) for 16h. The supernatants were assessed for IL-6 (E) and TNF (F) by ELISA.
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Figure 2: Serum-mediated increase of B. Burgdorferi phagocytosis is CR3 dependent. (A) CHO-CR3 (bottom panel) and CHO-CR4 (top panel) cells were incubated with Bb914 (m.o.i. = 50) in the presence of HI (blue histogram) or NMS (red histogram). After 6 h, the binding of B. burgdorferi was determined by flow cytometry. (B) RAW cells were incubated with Bb914 in the presence of HI (blue histogram), NMS (red histogram) or NMS + 10 μg/mL of a blocking CD11b antibody (green histogram). After 2 h, the cells were analyzed by flow cytometry. (C) CD11b-deficient BMMs were incubated with Bb914 in the presence of HI (blue histogram) or NMS (red histogram). The cells were analyzed by flow cytometry after 4 h. The grey histograms represent a 4 ºC control. The data represent one of at least 3 independent experiments performed in triplicate. (D) BMMs from C57Bl/6 mice were stimulated with live B. Burgdorferi (m.o.i. = 25) in the presence of NMS (blue bar) or C3-deficient serum (red bar). TNF levels in the stimulation supernatants were measured 16 h later by ELISA. (E, F) BMMs were stimulated with B. burgdorferi (m.o.i. = 25) or LPS (100 ng/mL) in the presence of HI (green bar) or NMS (blue bar) for 16h. The supernatants were assessed for IL-6 (E) and TNF (F) by ELISA.
Mentions: To demonstrate that the enhanced phagocytosis of B. burgdorferi in the presence of NMS was specific to CR3, we used CHO cells transfected with human CR3 (CD11b/CD18; CHO-CR3) or CR4 (CD11c/CD18; CHO-CR4) as controls 1, 7. Incubation of CHO-CR3, but not CHO-CR4, cells resulted in increased binding of the spirochete (Fig. 2A). Furthermore, the presence of a blocking CD11b antibody (M1/70) 1 also resulted in the reduction of B. burgdorferi phagocytosis in the presence of NMS in RAW cells (Fig. 2B). We also used BMMs generated from CD11b KO mice. Consistently, the presence of NMS during phagocytosis failed to increase the phagocytosis of B. burgdorferi in the absence of CD11b, compared to the use of HI serum (Fig. 2C), demonstrating that sera increased the phagocytosis of B. burgdorferi through its interaction with CR3.

View Article: PubMed Central - PubMed

Affiliation: 1. Department of Veterinary and Animal Sciences, University of Massachusetts Amherst, 01003 Amherst, MA, USA.

AUTOMATICALLY GENERATED EXCERPT
Please rate it.

Incubation of CHO-CR3, but not CHO-CR4, cells resulted in increased binding of the spirochete (Fig. 2A)... Furthermore, the presence of a blocking CD11b antibody (M1/70) also resulted in the reduction of B. burgdorferi phagocytosis in the presence of NMS in RAW cells (Fig. 2B)... Consistently, the presence of NMS during phagocytosis failed to increase the phagocytosis of B. burgdorferi in the absence of CD11b, compared to the use of HI serum (Fig. 2C), demonstrating that sera increased the phagocytosis of B. burgdorferi through its interaction with CR3... We argued that the CR3-dependent boost in phagocytosis in the presence of NMS would also reduce the production of TNF in response to B. burgdorferi... We stimulated BMMs with live B. burgdorferi at an m.o.i. of 25 for 16 h in the presence of NMS or C3-deficient serum... The presence of NMS significantly reduced the production of TNF by BMMs (Fig. 2D)... Importantly, the absence of C3 abrogated this inhibitory effect (Fig. 2D)... The effect of active serum on macrophages was specific for TNF, since the levels of IL-6 did not change in the presence of active serum (Fig. 2E)... Furthermore, the effect was specific of ligands that interact with CR3, since the presence of serum did not result in changes in the levels of TNF produced by BMMs in response to the TLR4 agonist, LPS (Fig. 2F)... Overall, these data demonstrate that CR3 is an anti-inflammatory phagocytic receptor both in the absence and the presence of the opsonin iC3b... In summary, our results show that serum C3-derived opsonins enhance the phagocytosis of B. burgdorferi mediated by CR3 further tempering the inflammatory output of macrophages induced by the integrin.

No MeSH data available.


Related in: MedlinePlus