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CD36 recruits α₅β₁ integrin to promote cytoadherence of P. falciparum-infected erythrocytes.

Davis SP, Lee K, Gillrie MR, Roa L, Amrein M, Ho M - PLoS Pathog. (2013)

Bottom Line: Using a combination of flow chamber assay, atomic force and confocal microscopy, we showed for the first time by loss- and gain-of function assays that in the resting state, the integrin α₅β₁ does not support adhesive interactions between IRBC and HDMEC.Clustering of β1 integrin is associated with an increase in IRBC recruitment as well as in adhesive strength after attachment (∼40% in both cases).Targeting integrins may provide a novel approach to decrease IRBC cytoadherence to microvascular endothelium.

View Article: PubMed Central - PubMed

Affiliation: Department of Microbiology, Immunology and Infectious Diseases, University of Calgary, Calgary, Alberta, Canada.

ABSTRACT
The adhesion of Plasmodium falciparum-infected erythrocytes (IRBC) to receptors on different host cells plays a divergent yet critical role in determining the progression and outcome of the infection. Based on our ex vivo studies with clinical parasite isolates from adult Thai patients, we have previously proposed a paradigm for IRBC cytoadherence under physiological shear stress that consists of a recruitment cascade mediated largely by P-selectin, ICAM-1 and CD36 on primary human dermal microvascular endothelium (HDMEC). In addition, we detected post-adhesion signaling events involving Src family kinases and the adaptor protein p130CAS in endothelial cells that lead to CD36 clustering and cytoskeletal rearrangement which enhance the magnitude of the adhesive strength, allowing adherent IRBC to withstand shear stress of up to 20 dynes/cm². In this study, we addressed whether CD36 supports IRBC adhesion as part of an assembly of membrane receptors. Using a combination of flow chamber assay, atomic force and confocal microscopy, we showed for the first time by loss- and gain-of function assays that in the resting state, the integrin α₅β₁ does not support adhesive interactions between IRBC and HDMEC. Upon IRBC adhesion to CD36, the integrin is recruited either passively as part of a molecular complex with CD36, or actively to the site of IRBC attachment through phosphorylation of Src family kinases, a process that is Ca²⁺-dependent. Clustering of β1 integrin is associated with an increase in IRBC recruitment as well as in adhesive strength after attachment (∼40% in both cases). The adhesion of IRBC to a multimolecular complex on the surface of endothelial cells could be of critical importance in enabling adherent IRBC to withstand the high shear stress in the microcirculations. Targeting integrins may provide a novel approach to decrease IRBC cytoadherence to microvascular endothelium.

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Inhibition of cytoadherence and adhesive strength on HDMEC transfected with small interference RNA of β1 integrin.(A) Western blot analysis of HDMEC lysates 72 h after transfection with 20 nM of negative siRNA and siRNA for β1 integrin ‘B’ and ‘D’, and CD36. Blots were probed with mAb anti-β1 integrin TS2/16 (top) and anti-α-tubulin (bottom). Results shown are representative of 3 independent experiments. (B) Densitometric analysis showing the effectiveness of the knockdown of β1 integrin (n = 3). (C) Adhesion of IRBC to β1 integrin and CD36 knock down endothelial monolayers (n = 3). (D) Force of detachment for IRBC on β1 integrin and CD36 knock down endothelial monolayers (n = 4).
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ppat-1003590-g004: Inhibition of cytoadherence and adhesive strength on HDMEC transfected with small interference RNA of β1 integrin.(A) Western blot analysis of HDMEC lysates 72 h after transfection with 20 nM of negative siRNA and siRNA for β1 integrin ‘B’ and ‘D’, and CD36. Blots were probed with mAb anti-β1 integrin TS2/16 (top) and anti-α-tubulin (bottom). Results shown are representative of 3 independent experiments. (B) Densitometric analysis showing the effectiveness of the knockdown of β1 integrin (n = 3). (C) Adhesion of IRBC to β1 integrin and CD36 knock down endothelial monolayers (n = 3). (D) Force of detachment for IRBC on β1 integrin and CD36 knock down endothelial monolayers (n = 4).

Mentions: To more specifically assess the role of β1 integrin in the increase in IRBC recruitment and the adhesive strength of the IRBC-integrin interaction, we performed gene knock down of β1 integrin in HDMEC by siRNA. Loss of β1 integrin protein production was confirmed by Western blot (Figure 4A and B). The targeted deletion of β1 integrin did not alter endothelial CD36 or ICAM-1 expression (data not shown). The loss of β1 integrin led to a reduction in IRBC adhesion in the flow chamber assay (Figure 4C) as well as the adhesive strength as measured by AFM (Figure 4D). These results confirmed a functional role for β1 integrin in mediating IRBC cytoadherence. Interestingly, when the α5 integrin was similarly knocked down (Figure 5A and B), a reduction in IRBC adhesion in the flow chamber assay was observed (Figure 5C) while adhesive strength by AFM was unaffected (Figure 5D). The lack of effect of α5 knock down on adhesive force was consistent with the inability of the mAb JBS5 to inhibit adhesive force (Figure 5E).


CD36 recruits α₅β₁ integrin to promote cytoadherence of P. falciparum-infected erythrocytes.

Davis SP, Lee K, Gillrie MR, Roa L, Amrein M, Ho M - PLoS Pathog. (2013)

Inhibition of cytoadherence and adhesive strength on HDMEC transfected with small interference RNA of β1 integrin.(A) Western blot analysis of HDMEC lysates 72 h after transfection with 20 nM of negative siRNA and siRNA for β1 integrin ‘B’ and ‘D’, and CD36. Blots were probed with mAb anti-β1 integrin TS2/16 (top) and anti-α-tubulin (bottom). Results shown are representative of 3 independent experiments. (B) Densitometric analysis showing the effectiveness of the knockdown of β1 integrin (n = 3). (C) Adhesion of IRBC to β1 integrin and CD36 knock down endothelial monolayers (n = 3). (D) Force of detachment for IRBC on β1 integrin and CD36 knock down endothelial monolayers (n = 4).
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC3757042&req=5

ppat-1003590-g004: Inhibition of cytoadherence and adhesive strength on HDMEC transfected with small interference RNA of β1 integrin.(A) Western blot analysis of HDMEC lysates 72 h after transfection with 20 nM of negative siRNA and siRNA for β1 integrin ‘B’ and ‘D’, and CD36. Blots were probed with mAb anti-β1 integrin TS2/16 (top) and anti-α-tubulin (bottom). Results shown are representative of 3 independent experiments. (B) Densitometric analysis showing the effectiveness of the knockdown of β1 integrin (n = 3). (C) Adhesion of IRBC to β1 integrin and CD36 knock down endothelial monolayers (n = 3). (D) Force of detachment for IRBC on β1 integrin and CD36 knock down endothelial monolayers (n = 4).
Mentions: To more specifically assess the role of β1 integrin in the increase in IRBC recruitment and the adhesive strength of the IRBC-integrin interaction, we performed gene knock down of β1 integrin in HDMEC by siRNA. Loss of β1 integrin protein production was confirmed by Western blot (Figure 4A and B). The targeted deletion of β1 integrin did not alter endothelial CD36 or ICAM-1 expression (data not shown). The loss of β1 integrin led to a reduction in IRBC adhesion in the flow chamber assay (Figure 4C) as well as the adhesive strength as measured by AFM (Figure 4D). These results confirmed a functional role for β1 integrin in mediating IRBC cytoadherence. Interestingly, when the α5 integrin was similarly knocked down (Figure 5A and B), a reduction in IRBC adhesion in the flow chamber assay was observed (Figure 5C) while adhesive strength by AFM was unaffected (Figure 5D). The lack of effect of α5 knock down on adhesive force was consistent with the inability of the mAb JBS5 to inhibit adhesive force (Figure 5E).

Bottom Line: Using a combination of flow chamber assay, atomic force and confocal microscopy, we showed for the first time by loss- and gain-of function assays that in the resting state, the integrin α₅β₁ does not support adhesive interactions between IRBC and HDMEC.Clustering of β1 integrin is associated with an increase in IRBC recruitment as well as in adhesive strength after attachment (∼40% in both cases).Targeting integrins may provide a novel approach to decrease IRBC cytoadherence to microvascular endothelium.

View Article: PubMed Central - PubMed

Affiliation: Department of Microbiology, Immunology and Infectious Diseases, University of Calgary, Calgary, Alberta, Canada.

ABSTRACT
The adhesion of Plasmodium falciparum-infected erythrocytes (IRBC) to receptors on different host cells plays a divergent yet critical role in determining the progression and outcome of the infection. Based on our ex vivo studies with clinical parasite isolates from adult Thai patients, we have previously proposed a paradigm for IRBC cytoadherence under physiological shear stress that consists of a recruitment cascade mediated largely by P-selectin, ICAM-1 and CD36 on primary human dermal microvascular endothelium (HDMEC). In addition, we detected post-adhesion signaling events involving Src family kinases and the adaptor protein p130CAS in endothelial cells that lead to CD36 clustering and cytoskeletal rearrangement which enhance the magnitude of the adhesive strength, allowing adherent IRBC to withstand shear stress of up to 20 dynes/cm². In this study, we addressed whether CD36 supports IRBC adhesion as part of an assembly of membrane receptors. Using a combination of flow chamber assay, atomic force and confocal microscopy, we showed for the first time by loss- and gain-of function assays that in the resting state, the integrin α₅β₁ does not support adhesive interactions between IRBC and HDMEC. Upon IRBC adhesion to CD36, the integrin is recruited either passively as part of a molecular complex with CD36, or actively to the site of IRBC attachment through phosphorylation of Src family kinases, a process that is Ca²⁺-dependent. Clustering of β1 integrin is associated with an increase in IRBC recruitment as well as in adhesive strength after attachment (∼40% in both cases). The adhesion of IRBC to a multimolecular complex on the surface of endothelial cells could be of critical importance in enabling adherent IRBC to withstand the high shear stress in the microcirculations. Targeting integrins may provide a novel approach to decrease IRBC cytoadherence to microvascular endothelium.

Show MeSH
Related in: MedlinePlus