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Modulating proximal cell signaling by targeting Btk ameliorates humoral autoimmunity and end-organ disease in murine lupus.

Hutcheson J, Vanarsa K, Bashmakov A, Grewal S, Sajitharan D, Chang BY, Buggy JJ, Zhou XJ, Du Y, Satterthwaite AB, Mohan C - Arthritis Res. Ther. (2012)

Bottom Line: Bruton's tyrosine kinase (Btk) is a proximal transducer of the BCR signal that allows for B-cell activation and differentiation.The effect of PCI-32765 on specific cell types was also investigated.These findings suggest that partial crippling of cell signaling in B cells and antigen presenting cells (APCs) may be a viable alternative to total depletion of these cells as a therapeutic modality for lupus.

View Article: PubMed Central - HTML - PubMed

ABSTRACT

Introduction: Systemic lupus erythematosus is a chronic autoimmune disease characterized by an abundance of autoantibodies against nuclear antigens. Bruton's tyrosine kinase (Btk) is a proximal transducer of the BCR signal that allows for B-cell activation and differentiation. Recently, selective inhibition of Btk by PCI-32765 has shown promise in limiting activity of multiple cells types in various models of cancer and autoimmunity. The aim of this study was to determine the effect of Btk inhibition by PCI-32765 on the development of lupus in lupus-prone B6.Sle1 and B6.Sle1.Sle3 mice.

Methods: B6.Sle1 or B6.Sle1.Sle3 mice received drinking water containing either the Btk inhibitor PCI-32765 or vehicle for 56 days. Following treatment, mice were examined for clinical and pathological characteristics of lupus. The effect of PCI-32765 on specific cell types was also investigated.

Results: In this study, we report that Btk inhibition dampens humoral autoimmunity in B6.Sle1 monocongenic mice. Moreover, in B6.Sle1.Sle3 bicongenic mice that are prone to severe lupus, Btk inhibition also dampens humoral and cellular autoimmunity, as well as lupus nephritis.

Conclusions: These findings suggest that partial crippling of cell signaling in B cells and antigen presenting cells (APCs) may be a viable alternative to total depletion of these cells as a therapeutic modality for lupus.

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Inhibition of Bruton's tyrosine kinase (Btk) by PCI-32765. (A) Fluorescent gel scan of splenic lysates from mice that had been treated with PCI-32765 or vehicle. Cells were incubated with the affinity probe PCI-33380, a fluorescently tagged derivative of PCI-32765, prior to lysis and visualization by SDS-PAGE. Binding of PCI-33380 indicates unbound Btk was available in the cells. The arrow indicates the predominant band labeled by the probe (at approximately 76 kDa, the expected molecular weight of Btk). The percentage of occupancy is based on densitometry relative to the vehicle-treated samples. (B) Total Btk expression was determined by western blot.
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Figure 3: Inhibition of Bruton's tyrosine kinase (Btk) by PCI-32765. (A) Fluorescent gel scan of splenic lysates from mice that had been treated with PCI-32765 or vehicle. Cells were incubated with the affinity probe PCI-33380, a fluorescently tagged derivative of PCI-32765, prior to lysis and visualization by SDS-PAGE. Binding of PCI-33380 indicates unbound Btk was available in the cells. The arrow indicates the predominant band labeled by the probe (at approximately 76 kDa, the expected molecular weight of Btk). The percentage of occupancy is based on densitometry relative to the vehicle-treated samples. (B) Total Btk expression was determined by western blot.

Mentions: Though Sle1 by itself is not sufficient for full-blown lupus to ensue, bicongenics bearing both Sle1 and Sle3 develop lupus nephritis, likely as a result of a cumulative impact on multiple cell types including both B cells and APCs [33]. Given that the B6.Sle1.Sle3 mouse strain develops more severe disease with spontaneous glomerulonephritis around 6 months of age, we next treated 4-month-old (pre-disease) B6.Sle1.Sle3 mice for 2 months with either PCI-32765 (n = 9) or a vehicle (n = 8) to determine if disease progression could be delayed or halted. Following treatment, splenocytes from the mice were isolated and assayed to ensure that PCI-32765 was binding Btk as previously reported (Figure 3A) [17]. Mice that had been treated with the vehicle displayed a thick band representative of unbound Btk. However, B6.Sle1.Sle3 mice that received drinking water containing PCI-32765 had significantly lighter bands, suggesting that Btk had been bound by PCI-32765 in these mice. We determined by densitometry that an average of 78.4% of the Btk was bound by PCI-32765 in the treatment group compared to the vehicle-treated mice (Figure 3A). The total expression of Btk was not affected in the PCI-32765-treated mice (Figure 3B).


Modulating proximal cell signaling by targeting Btk ameliorates humoral autoimmunity and end-organ disease in murine lupus.

Hutcheson J, Vanarsa K, Bashmakov A, Grewal S, Sajitharan D, Chang BY, Buggy JJ, Zhou XJ, Du Y, Satterthwaite AB, Mohan C - Arthritis Res. Ther. (2012)

Inhibition of Bruton's tyrosine kinase (Btk) by PCI-32765. (A) Fluorescent gel scan of splenic lysates from mice that had been treated with PCI-32765 or vehicle. Cells were incubated with the affinity probe PCI-33380, a fluorescently tagged derivative of PCI-32765, prior to lysis and visualization by SDS-PAGE. Binding of PCI-33380 indicates unbound Btk was available in the cells. The arrow indicates the predominant band labeled by the probe (at approximately 76 kDa, the expected molecular weight of Btk). The percentage of occupancy is based on densitometry relative to the vehicle-treated samples. (B) Total Btk expression was determined by western blot.
© Copyright Policy - open-access
Related In: Results  -  Collection

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Show All Figures
getmorefigures.php?uid=PMC3674619&req=5

Figure 3: Inhibition of Bruton's tyrosine kinase (Btk) by PCI-32765. (A) Fluorescent gel scan of splenic lysates from mice that had been treated with PCI-32765 or vehicle. Cells were incubated with the affinity probe PCI-33380, a fluorescently tagged derivative of PCI-32765, prior to lysis and visualization by SDS-PAGE. Binding of PCI-33380 indicates unbound Btk was available in the cells. The arrow indicates the predominant band labeled by the probe (at approximately 76 kDa, the expected molecular weight of Btk). The percentage of occupancy is based on densitometry relative to the vehicle-treated samples. (B) Total Btk expression was determined by western blot.
Mentions: Though Sle1 by itself is not sufficient for full-blown lupus to ensue, bicongenics bearing both Sle1 and Sle3 develop lupus nephritis, likely as a result of a cumulative impact on multiple cell types including both B cells and APCs [33]. Given that the B6.Sle1.Sle3 mouse strain develops more severe disease with spontaneous glomerulonephritis around 6 months of age, we next treated 4-month-old (pre-disease) B6.Sle1.Sle3 mice for 2 months with either PCI-32765 (n = 9) or a vehicle (n = 8) to determine if disease progression could be delayed or halted. Following treatment, splenocytes from the mice were isolated and assayed to ensure that PCI-32765 was binding Btk as previously reported (Figure 3A) [17]. Mice that had been treated with the vehicle displayed a thick band representative of unbound Btk. However, B6.Sle1.Sle3 mice that received drinking water containing PCI-32765 had significantly lighter bands, suggesting that Btk had been bound by PCI-32765 in these mice. We determined by densitometry that an average of 78.4% of the Btk was bound by PCI-32765 in the treatment group compared to the vehicle-treated mice (Figure 3A). The total expression of Btk was not affected in the PCI-32765-treated mice (Figure 3B).

Bottom Line: Bruton's tyrosine kinase (Btk) is a proximal transducer of the BCR signal that allows for B-cell activation and differentiation.The effect of PCI-32765 on specific cell types was also investigated.These findings suggest that partial crippling of cell signaling in B cells and antigen presenting cells (APCs) may be a viable alternative to total depletion of these cells as a therapeutic modality for lupus.

View Article: PubMed Central - HTML - PubMed

ABSTRACT

Introduction: Systemic lupus erythematosus is a chronic autoimmune disease characterized by an abundance of autoantibodies against nuclear antigens. Bruton's tyrosine kinase (Btk) is a proximal transducer of the BCR signal that allows for B-cell activation and differentiation. Recently, selective inhibition of Btk by PCI-32765 has shown promise in limiting activity of multiple cells types in various models of cancer and autoimmunity. The aim of this study was to determine the effect of Btk inhibition by PCI-32765 on the development of lupus in lupus-prone B6.Sle1 and B6.Sle1.Sle3 mice.

Methods: B6.Sle1 or B6.Sle1.Sle3 mice received drinking water containing either the Btk inhibitor PCI-32765 or vehicle for 56 days. Following treatment, mice were examined for clinical and pathological characteristics of lupus. The effect of PCI-32765 on specific cell types was also investigated.

Results: In this study, we report that Btk inhibition dampens humoral autoimmunity in B6.Sle1 monocongenic mice. Moreover, in B6.Sle1.Sle3 bicongenic mice that are prone to severe lupus, Btk inhibition also dampens humoral and cellular autoimmunity, as well as lupus nephritis.

Conclusions: These findings suggest that partial crippling of cell signaling in B cells and antigen presenting cells (APCs) may be a viable alternative to total depletion of these cells as a therapeutic modality for lupus.

Show MeSH
Related in: MedlinePlus