Anti-serpin antibody-mediated regulation of proteases in autoimmune diabetes

Abstract

Secretion of anti-serpin B13 autoantibodies in young diabetes-prone nonobese diabetic mice is associated with reduced inflammation in pancreatic islets and a slower progression to autoimmune diabetes. Injection of these mice with a monoclonal antibody (mAb) against serpin B13 also leads to fewer inflammatory cells in the islets and more rapid recovery from recent-onset diabetes. The exact mechanism by which anti-serpin activity is protective remains unclear. We found that serpin B13 is expressed in the exocrine component of the mouse pancreas, including the ductal cells. We also found that anti-serpin B13 mAb blocked the inhibitory activity of serpin B13, thereby allowing partial preservation of the function of its target protease. Consistent with the hypothesis that anti-clade B serpin activity blocks the serpin from binding, exposure to exogenous anti-serpin B13 mAb or endogenous anti-serpin B13 autoantibodies resulted in cleavage of the surface molecules CD4 and CD19 in lymphocytes that accumulated in the pancreatic islets and pancreatic lymph nodes but not in the inguinal lymph nodes. This cleavage was inhibited by an E64 protease inhibitor. Consequently, T cells with the truncated form of CD4 secreted reduced levels of interferon-γ. We conclude that anti-serpin antibodies prevent serpin B13 from neutralizing proteases, thereby impairing leukocyte function and reducing the severity of autoimmune inflammation.

FIGURE 1.

Expression of serpin B13 in the pancreas. Shown is the costaining of frozen pancreatic sections obtained from 6-week-old NOD mouse with anti-serpin B13 mAb and antibodies directed against glucagon (A), CD31 (B), and keratin-19 (C). Staining with the isotype control IgG2b failed to produce the pattern seen with anti-serpin B13 mAb (A, left panel). D, time course of serpin B13 expression at a young age. Pancreases from 4-, 14-, and 21-day-old NOD mice were stained with mAbs against serpin B13 (upper panels) and keratin-19 (lower panels). Scale bars = 50 μm (A and D) and 100 μm (B and C).

FIGURE 2.

Effect of anti-serpin B13 mAb on protease targets. A, serum-binding activity of serpin B13 in NOD mice isolated from different sources. Left panel, serum samples that were positive (S1–S3; n = 3) or negative (S4–S6; n = 3) for binding to serpin B13 produced in 293T cells were analyzed to determine the binding characteristics of this molecule produced in insect cells and E. coli as indicated. The assay was performed exactly as described previously (15), and data are expressed as -fold induction and represent the total -fold induction minus the -fold induction due to serum-binding activity in the presence of beads precoated with a control lysate (293T cells transfected with GFP). Luminex beads were loaded with 10–20 μg of purified protein or 1–2 ml of cell lysates. Right panel, Western blot analysis of purified proteins (0.5 μg of protein/lane in the first and second lanes) or cell lysates corresponding to 293T cells transfected with serpin B13 or GFP (50 μl of cell lysate/lane in the third and fourth lanes) that were used to perform the serum-binding activity assay depicted in the left panel. The blot was stained with anti-His₆ polyclonal antibody. B, left panel, cleavage of the (carboxybenzyl-Phe-Arg)₂-rhodamine 110 ((CBZ-Phe-Arg)₂-R110) substrate by cathepsin L (Cat L) in the presence of serpin B13 produced in insects and anti-serpin B13 mAb as indicated. Data are expressed as -fold induction of maximum fluorescence over the background (which was measured in the presence of the substrate alone). The average of three experiments described is shown. Right panel, Western blot analysis of serpin B13 staining with mAb raised against mouse serpin B13. The lysates of 293T cells transfected with either an empty vector (lane 1) or mouse serpin B13 (lane 2) were analyzed. N.S., not significant. C, in vivo effect of anti-serpin B13 mAb on cathepsin L in the pancreases of female NOD mice treated with anti-serpin B13 mAb (n = 4) or control IgG (n = 4). Four-week-old animals were injected four times intravenously (100 μg/injection) over a 10-day period. Representative analysis is depicted in the left panel, and results from three independent experiments are summarized in the right panel. The error bars indicate S.D. FU, fluorescence units.

FIGURE 3.

Effect of anti-serpin B13 mAb on CD4 and CD19 in pancreas-associated lymphocytes. A, left panels, FACS analysis of CD4 expression in the islets and inguinal lymph nodes of NOD mice treated with control IgG (n = 4), anti-serpin B13 mAb (n = 4), the E64 protease inhibitor (n = 4), or both anti-serpin B13 and the E64 inhibitor (n = 4). Over a 10-day period, 4-week-old mice (prescreened for the low levels of anti-serpin autoantibodies) were injected four times intravenously with mAb or control IgG (100 μg/injection) and 10 times intraperitoneally with E64 (10 mg/kg) or diluent as indicated. Animals were killed, and cell suspensions obtained from their organs were stained with PE-conjugated anti-CD4 mAb at 1:800. This dilution of anti-CD4 mAb allowed us to distinguish between high (M1) and low (M2) rates of expression of CD4 in T cells. Right panels, the average of three experiments is shown. Data are expressed as the ratio between populations with low and high CD4 expression. B, left panels, FACS analysis of B220 and CD19 expression in the pancreatic and inguinal lymph nodes of NOD mice treated with anti-serpin B13 mAb and/or E64 (n = 8) exactly as described for A. Animals were killed, and their lymph nodes were stained with PE-conjugated anti-B220 mAb (1:200) and FITC-conjugated anti-CD19 mAb (1:100). The M1 and M2 regions depict high and low CD19 expressers, respectively. Right panels, the average of three experiments described is shown. Data are expressed as the ratio between populations with low versus high rates of CD19 expression. DMSO, dimethyl sulfoxide.

FIGURE 4.

Effect of anti-serpin B13 natural autoantibodies on CD4 in the pancreas-associated lymphocytes. Left panels, analysis of CD4 expression in 4-week-old female NOD mice that had been prescreened for low (SBA^low) or high (SBA^high) secretion of anti-serpin B13 autoantibodies and received either E64 or diluent (PBS) for 10 consecutive days exactly as described in the legend to Fig. 3. The animals were killed, and cell suspensions obtained from their organs were stained with PE-conjugated anti-CD4 mAb at 1:800. This dilution of anti-CD4 mAb allowed us to distinguish between high (M1) and low (M2) rates of expression of CD4 in T cells. Each histogram was generated by examining four animals. Right panels, the average of three experiments described is shown. The data are expressed as the ratio between populations with low versus high rates of CD4 expression. The errors bars indicate S.D. DMSO, dimethyl sulfoxide.

FIGURE 5.

Cleavage of CD4 and CD19 in the PLNs. Shown are the results from Western blot analysis of CD4 and CD19 in cells from inguinal lymph nodes (ILNs) and PLNs, sorted for the high (R1 and R2) and intermediate/low (R3) levels of these markers. The blots were stained with an anti-CD19 antibody that recognizes the cytoplasmic portion of the molecule (A) or two different anti-CD4 antibodies that recognize the intracellular (C-18) or extracellular (J15) portion of CD4 (B). The control blot (A, right panels) was stained with the secondary reagent only. The data are representative of three independent experiments.

FIGURE 6.

Diminished secretion of IFN-γ in T cells with a cleaved form of the CD4 (CD4^low) molecule. A, CD4^high and CD4^low T cells were isolated from the PLNs of BDC2.5 TCR transgenic NOD mice by sorting cells that positively stained with FITC-conjugated anti-Vβ4 TCR chain mAb (1:500) and PE-conjugated anti-CD4 mAb (1:800). The cells were then stimulated with different concentrations of the BDC2.5 mimotope in the presence of antigen-presenting cells (left panel) or phorbol esters (phorbol 12-myristate 13-acetate (PMA)) and ionomycin (right panel). At 48 h after initiation of stimulation, the cells were counted, and culture supernatants were examined by ELISA for IFN-γ concentration. The average of three independent experiments described is shown. B, after stimulation for 72 h with the BDC2.5 mimotope-pulsed antigen-presenting cells, the cells were rested for 48 h and then restimulated with phorbol 12-myristate 13-acetate (10 ng/ml) and ionomycin (1 μm). The GolgiStop reagent was added during the last 8 h of secondary culture. The cytokine production was examined by intracellular staining of cells with allophycocyanin-conjugated mAb against IFN-γ (1:100). The data are representative of three independent experiments. The error bars indicate S.D.