Cutting Edge: Diabetes-Associated Quantitative Trait Locus, Idd4, Is Responsible for the IL-12p40 Overexpression Defect in Nonobese Diabetic (NOD) Mice

Pedro B. Simpson, Monica S. Mistry, Richard A. Maki, Weidong Yang, David A. Schwarz, Eric B. Johnson, Francisco M. Lio and David G. Alleva
J Immunol October 1, 2003, 171 (7) 3333-3337; DOI: https://doi.org/10.4049/jimmunol.171.7.3333

Article Figures & Data

Figures

  • FIGURE 1.
    FIGURE 1.

    Elevated production of IL-12p40 by Mφ from the NOD strain. Thioglycollate-elicited peritoneal Mφ (2 × 105) from five control strains (BALB/c, A/J, C57BL/6 (B6), C57BL/10 (B10), and C3H/OuJ) and the diabetes-prone NOD strain were activated with LPS (100 ng/ml) and incubated for 24 h (A) and from 24 to 72 h (B). (At the end of 24 h, conditioned medium was removed and cultures were replenished with fresh medium and LPS for an additional 48 h.) Total IL-12p40 levels were measured using an ELISA. Shown are means ± SEM of several experiments per strain (N denoted in parentheses) of relative IL-12p40 levels from each strain normalized to B6 levels which were given the value of 1. The mean ± SEM of B6 Mφ IL-12p40 levels from several experiments (n = 8) was 3.4 ± 0.9 ng/ml for 24-h cultures, and 0.6 ± 0.1 ng/ml for 24- to 72-h cultures.

  • FIGURE 2.
    FIGURE 2.

    Elevated production of IL-12p40 by Mφ from the B6.NODIdd4 and B6.NODIdd1/Idd4 congenic strains. Thioglycollate-elicited peritoneal Mφ (2 × 105) from seven B6.NODIdd-congenic strains containing single (Idd1, Idd4, Idd13), double (Idd1/4, Idd1/5, Idd1/6, Idd1/7) or triple (Idd1/3/10) Idd loci on a C57BL/6 background, and from the normal C57BL/6 and diabetes-prone NOD control strains were activated with LPS (100 ng/ml) and incubated for 24 h. Conditioned medium was collected and IL-12p40 levels were measured by ELISA. Shown are means ± SEM of several experiments per strain (N denoted in parentheses) of relative IL-12p40 levels from each strain normalized to those of the NOD which were given the value of 1. ∗, Significantly (p < 0.05) different from C57BL/6 mean values. The mean ± SEM of NOD Mφ IL-12p40 levels from several experiments (n = 11) was 14.8 ± 2.3 ng/ml for 24-h cultures.

  • FIGURE 3.
    FIGURE 3.

    Elevated expression of IL-12p40 mRNA by the B6.NODIdd4 and B6.NODIdd1/Idd4 congenic strains. Thioglycollate-elicited peritoneal Mφ (107) from C57BL/6, NOD, B6.NOD Idd4, and B6.NOD Idd1/Idd4 strains were activated with LPS (100 ng/ml) for 4 and 16 h. Total RNA was extracted and mRNA levels of IL-12p40 and IL-12p35 subunits were assessed in an RNase protection assay. Samples were separated on a sequencing gel and visualized by autoradiography. The intensities of each IL-12p40 and p35 mRNA band were normalized to the loading control L32 mRNA band via densitometry analysis using the ImagePro software. Results represent one of at least three experiments that had similar results.

  • FIGURE 4.
    FIGURE 4.

    Elevated production of IL-12p40 by a CBA-NOD recombinant congenic strain containing the Idd4 locus. Thioglycollate-elicited peritoneal Mφ (2 × 105) from four CBA-NOD recombinant congenic strains (CBcNO6, CBcNO7-C, CBcNO7-D, and NOcCB-1), each containing a different set of Idd loci, were activated with LPS (100 ng/ml) and incubated for 24 h. Conditioned medium was collected and IL-12p40 (A) and TNF-α (B) levels were measured by ELISA. Shown are mean ± SEM values of triplicate cultures from one of two experiments that had similar results. C, mRNA levels of 4-h LPS (100 ng/ml)-activated Mφ (107) from each strain that were assessed using the RNase protection assay. The intensities of each IL-12p40 and p35 mRNA band were normalized to the loading control L32 mRNA band via densitometry analysis using the ImagePro software.

Tables

  • Table I.

    Genotypic characterization of the IL-12b allele and Idd4 intervals in recombinant congenic murine strains

    StrainaIL-12p40 AllelebIL-12p40 Expression DefectIdd4 Locusc
    506 nt880 ntInterval (cM)Markers
    C57BL/6TT−NoneNone
    B6.NOD.Idd1TT−NoneNone
    B6.NOD.Idd1/3/10TT−NoneNone
    B6.NOD.Idd4CC+20–72D11Mit20−
    D11mit42
    B6.NOD.Idd1/Idd4CC+20–72D11Mit20−
    D11mit42
    B6.NOD.Idd1/Idd5TT−NoneNone
    B6.NOD.Idd1/Idd6TT−NoneNone
    B6.NOD.Idd13TT−NoneNone
    CBATT−NoneNone
    CBc.NO7CTT−40–48D11Acrb−
    D11Mit36
    CBc.NO6TT−40–70D11Acrb−
    D11Mit214
    NOcCB-1CC+17–40D11Mit84−
    D11Acrb
    NODCC+n.a.n.a.
    DBA/2CdCd−dn.a.n.a.

    • a Recombinant congenic strains with Idd4 loci on a C57BL/6 background (13 ) or with roughly 50% CBA and 50% NOD genomes with different combinations of Idd loci (12 ).

    • b Polymorphism in the IL-12p40 allele that distinguishes the NOD from the C57BL/6 and CBA alleles (14 ).

    • c Idd4 intervals on murine chromosome 11 defined by different microsatellite markers (12 ,13 ).

    • d Refs. 6 and 14 .

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