The CXCR4/CXCL12 (SDF-1) signalling pathway protects non-obese diabetic mouse from autoimmune diabetes

Abstract

Chemokines and their receptors are part of polarized T helper 1 (Th1)- and Th2-mediated immune responses which control trafficking of immunogenic cells to sites of inflammation. The chemokine stromal cell-derived factor-1 CXCL-12 (SDF-1) and its ligand the CXCR4 chemokine receptor are important regulatory elements. CXCR4 is expressed on the surface of CD4(+) T cells, dendritic cells and B lymphocytes. Levels of CXCR4 mRNA were increased in pancreatic lymph nodes (PLNs) of 4-week-old non-obese diabetic (NOD) mice in comparison to Balb/C mice. However, a significant reduction of CXCR4 was noticed at 12 weeks both at the mRNA and protein levels while expression increased in the inflamed islets. The percentage of SDF-1 attracted splenocytes in a transwell chemotaxis assay was significantly increased in NOD versus Balb/c mice. SDF-1 attracted T cells completely abolished the capacity of diabetogenic T cells to transfer diabetes in the recipients of an adoptive cell co-transfer. When T splenocytes from NOD females treated with AMD3100, a specific CXCR4 antagonist, were mixed with diabetogenic T cells during adoptive cell co-transfer experiments, prevalence of diabetes in the recipients rose from 33% to 75% (P < 0.001). This effect was associated with an increase of interferon (IFN)-gamma mRNA and a reduction of interleukin (IL)-4 mRNA levels both in PLNs and isolated islets. AMD3100 also reduced IL-4 and IL-10 production of plate-bound anti-CD3 and anti-CD28-stimulated splenocytes. Immunofluorescence studies indicated that AMD3100 reduced the number of CXCR4(+) and SDF-1 positive cells in the inflamed islets. We can conclude that the CXCL-12/CXCR4 pathway has protective effects against autoimmune diabetes.

Fig. 1

Percentages of CXCR4/cyclophilin mRNA levels in non-obese diabetic female mice (n = 4, black bars) in comparison to Balb/C mice (n = 4, open bars) at 4 and 12 weeks of age in pancreatic lymph nodes (a) or islets (b).

Fig. 2

Representative fluorescence activated cell sorter (FACS) analysis of pancreatic lymph nodes of non-obese diabetic female mice at 4 weeks (grey lines) or 12 weeks (dark lines) for the presence of CXCR4 (a), CXCR3 (b) and CCR5 (c).

Fig. 3

SDF-1 attracted CXCR4⁺ cells completely abolished the capacity of diabetogenic T cells to transfer diabetes (open circles) in contrast to cells from the upper chambers (closed circles). The incidence curve of mice transferred with diabetogenic T cells is illustrated as closed squares.

Fig. 4

In vivo administration of AMD3100 accelerates adoptive transfer of diabetes. The incidence curve for AMD3100-treated mice (closed circles) was significantly higher than sham-treated animals (open circles) (P < 0·05, Kaplan–Meier test).

Fig. 5

Immunofluorescence studies of pancreatic glands of 12-week-old non-obese diabetic mice treated with phosphate-buffered saline (PBS) (a,c) or 10 mg/kg AMD3100 a specific CXCR4 antagonist (b,d). AMD3100 reduced the number of CXCR4⁺ cells (green, a,b) in the islets and the number of SDF-1 positive cells surrounding the islets (red) while the number of infiltrating CD3⁺ cells (green, c,d) increased.

Fig. 6

In vitro studies of cell trafficking using double chamber culture plates using splenocytes of 4-week-old (a) or 12-week-old (b) mice. SDF-1 (black bars) increased significantly the number of transmigrated lymphocytes in non-obese diabetic mice versus Balb/c mice at 4 weeks and this effect was blocked by adding AMD3100 in the upper chamber (c). **P-value < 0·01.

Fig. 7

In vitro incubation with AMD3100 (closed bars) reduced significantly the levels of T helper 2 (Th2) cytokines produced by diabetogenic T cells in comparison to control (open bars) after plate-bound anti-CD3 and anti-CD28 stimulation in contrast to similar levels of interferon (IFN)-γ production.