Protein kinase Cβ is required for lupus development in Sle mice

Abstract

Objective: To evaluate the requirement for protein kinase Cβ (PKCβ) in the development of lupus in mice, and to explore the potential of targeting PKCβ as a therapeutic strategy in lupus.

Methods: Congenic mice bearing the disease loci Sle1 or Sle1 and Sle3, which represent different stages of severity in the development of lupus, were crossed with PKCβ-deficient mice. The effect of PKCβ deficiency in lupus development was analyzed. In addition, the effects of the PKCβ-specific inhibitor enzastaurin on the survival of B cells from mice with lupus and human 9G4-positive B cells as well as the in vivo effect of enzastaurin treatment on the development of lupus in Sle mice were investigated.

Results: In Sle mice, PKCβ deficiency abrogated lupus-associated phenotypes, including high autoantibody levels, proteinuria, and histologic features of lupus nephritis. Significant decreases in spleen size and in the peritoneal B-1 cell population, reduced numbers of activated CD4 T cells, and normalized CD4:CD8 ratios were observed. PKCβ deficiency induced an anergic B cell phenotype and preferentially inhibited autoreactive plasma cells and autoantibodies in mice with lupus. Inhibition of PKCβ enhanced apoptosis of both B cells from Sle mice and human autoreactive B cells (9G4 positive). Treatment of Sle mice with the PKCβ-specific inhibitor enzastaurin prevented the development of lupus.

Conclusion: This study identifies PKCβ as a central mediator of lupus pathogenesis, suggesting that PKCβ represents a promising therapeutic target for the treatment of systemic lupus erythematosus. Moreover, the results indicate the feasibility of using a PKCβ inhibitor for the treatment of lupus.

Figure 1

Protein kinase Cβ (PKCβ) deficiency prevents lupus development in Sle mice. A, Levels of serum IgG anti–double-stranded DNA (anti-dsDNA) and antihistone/anti-dsDNA autoantibodies from the indicated mouse strains (8–10 months old), as determined by enzyme-linked immunosorbent assay. For analysis of anti-dsDNA IgG and antihistone/anti-dsDNA, sera were diluted 100-fold and 200-fold, respectively. Values are the mean ± SD of ≥3 mice per group. B, Frozen kidney sections from 5-month-old mice, stained with fluorescein isothiocyanate–labeled anti-IgG2b or anti-IgG2c. C, Paraffin-embedded kidney sections from the same mice as in B, stained with periodic acid–Schiff reagent. Enlarged glomeruli that are typical of Sle1.Sle3 mice were not observed in PKCβ-deficient Sle mice. Images in B and C are representative of 2 independent experiments. D, Proteinuria levels, as measured with Uristix strips. Values are the mean ± SD of 8–10 mice per strain. * = P <0.05; ** = P < 0.01. In B and C, original magnification × 20 and × 40, respectively.

Figure 2

Protein kinase Cβ (PKCβ) deficiency reverses splenomegaly and restores the CD4:CD8 T cell ratio in Sle mice. A, Spleen weights in the indicated mouse strains at age 8 months. B, Representative results of flow cytometric analysis of spleen T cells from 5–6-month-old mice. Cells were stained with CD4 and CD8 antibodies. C and D, Absolute numbers of activated CD69+ cells in the CD4+ T cell population (C) and in the B220+ cell population (D). Results shown in B, C, and D were obtained from the same group of mice. Bars in A, C, and D show the mean ± SD of 5 mice per group (A) and 3 mice per group (C and D). * = P < 0.05; ** = P < 0.01.

Figure 3

Protein kinase Cβ (PKCβ) deficiency decreases the number of peritoneal B-1 cells in Sle mice. A, Flow cytometric analysis of peritoneal B-1 cells from the indicated mouse strains (5–6 months old). The cells were stained with B220 and CD5 antibodies. The encircled areas show B-1a cells. Results are representative of 3 independent experiments. B, Absolute numbers of B-1a (CD5+B220^low) and B-2 (B220+CD23+) cells in the peritonea of the indicated mouse strains. Bars show the mean ± SD of 3 independent experiments.

Figure 4

PKCβ deficiency results in a reduction in plasma cell numbers and abrogates spontaneous germinal cell (GC) formation in Sle mice. A, Flow cytometric analysis of splenocytes from 5–6-month-old PKCβ-deficient Sle mice. Cells were stained with the indicated antibodies. The encircled areas show spleen plasmablasts (CD138^highB220+) and plasma cells (CD138^highB220^low/negative). Results are representative of 3 independent experiments. B, Expression of IgG anti-dsDNA plasma cells (antibody-secreting cells [ASCs]) in 1 × 10⁶ cells from spleen or bone marrow, as determined by enzyme-linked immunospot assay. Bars show the mean ± SD. ** = P < 0.01. C, Representative immunofluorescence images of spontaneous GC (CD19+GL7+) formation in spleen sections. Spleen sections from the indicated mouse strains were stained with GL7 (green), CD3 (red), and CD19 (blue). Original magnification × 20. D, Representative results of flow cytometric analysis of GC cells (CD19+CD95+GL7+) in the indicated mouse strains. Results shown in A, B, and C were obtained from the same group of mice. See Figure 1 for other definitions.

Figure 5

Protein kinase Cβ (PKCβ) deficiency impairs the B cell receptor response and survival of B cells from Sle mice. A, In vitro responses of B cells to anti-IgM or lipopolysaccharide (LPS) stimulation. Splenocytes from 6-month-old mice were labeled with 5,6-carboxyfluorescein succinimidyl ester (CFSE) (shaded area) and stimulated with anti-IgM or LPS or were left in the medium without any added stimuli for 48 hours (black lines). Top, PKCβ deletion reduced the proliferative response of B cells to anti-IgM stimulation but not LPS stimulation. Bottom, PKCβ deficiency reduced up-regulation of CD69 and CD86 expression, as determined by fluorescence-activated cell sorting analysis. B, Effect of PKCβ deficiency on cell viability. Purified spleen B cells from the indicated mouse strains were incubated in RPMI 1640 medium. Cell viability was assayed by flow cytometry, using an annexin V detection kit. Bars show the mean ± SD. C, Expression of Bcl-x_L in anti-IgM–stimulated splenocytes from the indicated mouse strains, as analyzed by Western blotting. Actin was used as a loading control. D, Effect of PKCβ deletion on calcium signaling in B cells from Sle mice. Splenocytes from the indicated mouse strains were loaded with 1 μM Fura Red to evaluate their ability to flux Ca²⁺ in response to anti-IgM. B220+ cells were gated for the analysis. All data shown are representative of at least 2 independent experiments.

Figure 6

The protein kinase Cβ (PKCβ)–specific inhibitor enzastaurin induces apoptosis of lupus B cells and prevents lupus development in Sle mice. A, Effect of enzastaurin on apoptosis of lupus B cells. Purified splenic B cells were treated with anti-IgM antibody in the presence or absence (control) of enzastaurin for 48 hours and analyzed with an annexin V detection kit. The fractions of annexin V–positive (apoptotic) cells in the samples treated with only anti-IgM (control) are set at 1. B, Sensitivity of human 9G4-positive and 9G4-negative B cells to PKCβ inhibition. Purified splenic B cells were treated with enzastaurin for 24 hours. The apoptotic fractions from untreated samples are set at 1. Results are representative of 2 independent experiments. C, Levels of serum IgG anti–double-stranded DNA (anti-dsDNA) and antihistone/anti-dsDNA autoantibodies from vehicle-treated control mice and enzastaurin-treated mice, as analyzed by enzyme-linked immunosorbent assay. Bars in A–C show the mean ± SD of 3 independent experiments. D, Representative immunofluorescent images of IgG deposition (top) and glomeruli (bottom) in kidney sections from Sle1.Sle3 mice treated with vehicle or enzastaurin. Original magnification × 20 (top); × 40 (bottom). PAS = periodic acid–Schiff.