Anti-FcεR1 antibody injections activate basophils and mast cells and delay Type 1 diabetes onset in NOD mice

Abstract

Mounting evidence suggests that helminth infections protect against autoimmune diseases. As helminths cause chronic IgE-mediated activation of basophils and mast cells we hypothesized that continuous activation of these cells could prevent diabetes onset in nonobese diabetic (NOD) mice in the absence of infection. Anti-FcεR1 activated basophils and mast cells and resulted in the release of IL-4 and histamine into the bloodstream. Anti-FcεR1-treated NOD mice showed a type 2 shift in insulin-specific antibody production and exhibited significant delays in diabetes onset. IL-4 responses played a partial role as the protective effect of anti-FcεR1 therapy was diminished in IL-4-deficient NOD mice. In contrast, histamine signaling was not required as anti-FcεR1-mediated protection was not reduced in mice treated with histamine receptor blockers. These results demonstrate that anti-FcεR1 therapy delays diabetes onset in NOD mice and suggest that chronic basophil and mast cell activation may represent a new avenue of therapy for Th1-associated autoimmune diseases.

Figure 1. Anti-FcεR1 activates basophils and mast cells in vitro

(A) May-Grünwald staining of peritoneal mast cells after enrichment by density centrifugation. (B) Histamine release from mast cells in response to increasing concentrations of anti-FcεR1. (C) Representative dot plots of intracellular IL-4 staining of basophils after incubation with media or anti-FcεR1. Initial gating of lymphocytes and lower half of granulocyte region in FSC/SSC plot (left panel). Basophils identified as CD4-B220-IgE+ (middle panel). IL-4 staining of basophils after incubation with media (upper right panel) or anti-FcεR1 (lower right panel). (D) Percentages of basophils that stain positively for IL-4 after incubation with media or anti-FcεR1. (E) IL-4 release from whole blood after incubation with anti-FcεR1. Error bars denote SEM. Results from 4-6 animals per group are shown. Each experiment was independently performed twice. Statistical significance assessed per one-tailed paired T-test.

Figure 2. Effects of anti-FcεR1 injections in vivo

Percentages of circulating white blood cells that are basophils (A) and percentages of peritoneal cells that are mast cells (B) in mice one day after intraperitoneal injection of 50 μg of isotype control antibody or anti-FcεR1. (C) Plasma IL-4 concentration as measured by in vivo cytokine capture assay one day after anti-FcεR1 or isotype antibody injection. (D) Histamine concentration in plasma 30 minutes after anti-FcεR1 or isotype antibody injection. Statistical significance between groups was assessed by the Mann-Whitney test (*<0.05). Joined data from two independent experiments is shown with 5 animals per group and experiment.

Figure 3. Treatment with anti-FcεR1 delays the onset of diabetes

(A) Mean blood glucose levels and (B) percentages of NOD mice with diabetes during treatment with weekly injections of 50 μg anti-FcεR1 (n=9) or IgG control antibody (n=17). (C) Mean total numbers of pancreatic islets from anti-FcεR1 or isotype treated mice at 16 weeks of age (9-10 animals per group). Pancreatic islets were classified as non-infiltrated, periinsulitis, and intrainsulitis with less than or more than 50% infiltrated lymphocytes. (D) Blood glucose levels and (E) percentages of diabetic mice that received either weekly injections of 50 μg anti-FcεR1 for four weeks or injections of 25 or 50 μg anti-FcεR1 twice a week for four weeks (n=8 per group) or isotype controls (n=11). Error bars denote SEM. Statistical significance between groups was assessed by the Mann-Whitney test (*<0.05). Shown are the results from 2-3 independent experiments.

Figure 4. Cytokine and antibody response after 10 weeks of anti-FcεR1/isotype treatment at 16 weeks of age

(A) IFNγ, (B) IL-4, and (C) IL-5 cytokine production from spleen cells and (D) IFNγ and (E) IL-5 cytokine production from pancreatic lymph node cells in response to anti-CD3/anti-CD28. (F) Frequency of splenic CD4+IFNγ+ and (G) CD8+IFNγ+, as well as (H) CD4+IL-4+ and (I) CD4+IL-17+ T cells in response to anti-CD3/anti-CD28. (J) Plasma levels of insulin-specific IgG1 and (K) IgG2c. Statistical significance between groups was assessed by the Mann-Whitney test. Shown are the results from two independent experiments with a total of 9-10 animals per group.

Figure 5. Frequency of regulatory cell types and IL-10 production after 10 weeks of anti-FcεR1/isotype treatment at 16 weeks of age

(A) Frequency of splenic and (B) pancreatic lymph node CD4+CD25+FoxP3+ regulatory T cells in response to anti-CD3/anti-CD28. (C) Frequency of spontaneous splenic CD8+FoxP3+ regulatory T cells, (D) anti-CD3/anti-CD28 stimulated IL-10+ B-cells, and (E) B220+CD5+CD1d+ regulatory B-cells. (F) IL-10 release from spleen as well as (G) pancreatic lymph node cells in response to anti-CD3/anti-CD28. Statistical significance between groups was assessed by the Mann-Whitney test (spleen: 9-10 animals per group, pancreatic lymph node: 6 animals per group, CD8+ and B-regulatory cells: 4 animals per group). Shown are the results from two independent experiments.

Figure 6. Histamine receptor blockage does not decrease anti-FcεR1 mediated delay of diabetes onset

(A) Stomach pH from animals that were treated for 2 weeks with the histamine receptor blockers cimetidine and fexofenidine (5 animals per group). (B) Representative pictures of mice that were injected i.v. with toluidine blue following challenge with anti-FcεR1 (left ear) and IgG control (right ear). The bottom mouse was treated with fexofenidine, a histamine receptor 1 blocker, whereas the top mouse is an untreated control. (C) Blood glucose levels and (D) percentages of NOD mice with diabetes in the setting of continuous administration of histamine receptor blockers (anti-HR1/2) and treatment with weekly injections of 50 μg anti-FcεR1 or IgG control antibody. Shown are the results from two independent experiments with a total of 10 mice per group. Error bars denote SEM. Statistical significance between groups was assessed by the Mann-Whitney test (*<0.05).

Figure 7. Deficiency of IL-4 reduces anti-FcεR1 mediated protective effect

(A) Blood glucose levels and (B) percentages of mice with diabetes in IL-4 deficient NOD mice that were injected weekly with 50 μg anti-FcεR1 (n=11) or an IgG control antibody (n=15). Error bars denote SEM. Statistical significance between groups was assessed by the Mann-Whitney test (*<0.05). Shown are the results from four independent experiments.