Susceptibility quantitative trait loci for pathogenic leucocytosis in SCG/Kj mice, a spontaneously occurring crescentic glomerulonephritis and vasculitis model

Abstract

The spontaneous crescentic glomerulonephritis-forming/Kinjoh (SCG/Kj) mouse, a model of human crescentic glomerulonephritis (CrGN) and systemic vasculitis, is characterized by the production of myeloperoxidase-specific anti-neutrophil cytoplasmic autoantibody (MPO-ANCA) and marked leucocytosis. This study was performed to identify the specific populations of leucocytes associated with CrGN and susceptibility loci for pathogenic leucocytosis. Four hundred and twenty female (C57BL/6 × SCG/Kj) F2 intercross mice were subjected to serial flow cytometry examination of the peripheral blood (PB). Kidney granulocytes and monocytes were examined histopathologically. Linkage analyses were performed with 109 polymorphic microsatellite markers. Correlation studies revealed that increase of the granulocytes, F4/80(+) cells, CD3(+) CD4(-) CD8(-) T cells and dendritic cells (DCs) in peripheral blood (PB) were associated significantly with glomerulonephritis, crescent formation and vasculitis. In kidney sections, F4/80(low) cells were observed in crescent, while F4/80(high) cells were around the Bowman's capsules and in the interstitium. Numbers of F4/80(+) cells in crescents correlated significantly with F4/80(+) cell numbers in PB, but not with numbers of F4/80(+) cells in the interstitium. Genome-wide quantitative trait locus (QTL) mapping revealed three SCG/Kj-derived non-Fas QTLs for leucocytosis, two on chromosome 1 and one on chromosome 17. QTLs on chromosome 1 affected DCs, granulocytes and F4/80(+) cells, but QTL on chromosome 17 affected DCs and granulocytes. We found CrGN-associated leucocytes and susceptibility QTLs with their positional candidate genes. F4/80(+) cells in crescents are considered as recruited inflammatory macrophages. The results provide information for leucocytes to be targeted and genetic elements in CrGN and vasculitis.

Keywords: SCG/Kj mice; anti-neutrophil cytoplasmic autoantibody; crescentic glomerulonephritis; quantitative trait locus; vasculitis.

Fig. 1

Increased numbers of Gr-1⁺ granulocytes (a), F4/80⁺ macrophages/monocytes (b), dendritic cells (DCs) (c) and CD4⁻CD8⁻ T cells (d) in peripheral blood (PB) of [C57BL/6 × spontaneous crescentic glomerulonephritis-forming/Kinjoh (SCG/Kj)] F₂ intercross (BSF₂) mice with glomerulonephritis (GN). Not plasmacytoid (CD11c⁺B220⁺) but conventional (CD11c⁺B220⁻) DCs increased in GN (+) mice (e,f). Frequencies of each leucocyte in PB were counted using flow cytometry in C57BL/6 (B6), (C57BL/6 × SCG/Kj) F₁ (BSF₁), BSF₂ without GN [GN (−)] and BSF₂ with GN [GN (+)] mice at 12 weeks of age. Numbers of each leucocyte were calculated by multiplying the frequency and the whole white blood cell number. Whole leucocyte gates (blood cells except red blood cells and platelets) were examined. For CD4⁻CD8⁻ T cells, CD3⁺ cells were further gated and analysed. Mean values ± standard error of the mean are shown. P-values were determined with analysis of variance (anova) and Fisher's protected least significant difference procedure. Numbers of mice are shown in parentheses.

Fig. 2

Distribution of Gr-1⁺ cells, myeloperoxidase (MPO⁺) cells and F4/80⁺ cells in healthy and glomerulonephritic glomeruli with crescents [Cr (+)] and without crescents [Cr (−)]. Upper photographs: cortical glomeruli, lower photographs: interstitium. (a–c) Gr-1⁺ cells were detected mainly within the glomerular tuft (arrows). Gr-1⁺ cells were also detected in crescents to a lesser extent (arrowhead), whereas they were rarely observed in the peri-glomerular area and in the interstitium. (d–f) As with Gr-1⁺ cells, MPO⁺ cells were found mainly in the glomerular tuft (arrows). They were detected occasionally in crescents, while they were rarely found in the peri-glomerular area and in the interstitium. (g–i) F4/80⁺ cells were present in the medullary and cortical interstitium in healthy mice. In mice with non-crescentic [Cr (−)] GN, an increased presence of F4/80⁺ cells was demonstrated in the peri-glomerular area (arrow) and in the interstitium. This was the case in mice with crescentic [Cr (+)] GN to a greater extent, and F4/80⁺ cells were also detected in crescents. F4/80⁺ cells in the interstitium and peri-glomerular area were F4/80^high (arrows), while those in crescents were F4/80^low (arrowheads). Original magnifications × 200. Numbers of mice: healthy, one; Cr (−) GN, three; Cr (+) GN, eight.

Fig. 3

Distribution of F4/80⁺ cells in glomerulonephritic kidney: the difference between GN with crescents [Cr (+)] and without crescents [Cr (−)]. (a) More F4/80⁺ cells were demonstrated within crescents and peri-glomerular area in crescentic GN kidneys than in kidneys of GN without crescents. (b) F4/80⁺ cells were extensively present in the medullarly interstitium, especially in nephritic mice with crescents. Mean values ± standard error of the mean are shown are shown. Numbers of mice: Cr (−), four; Cr (+), eight.

Fig. 4

Numbers of F4/80⁺ cell in crescents presented positive correlation with F4/80⁺ cell numbers in peripheral blood at the onset of GN. Correlation coefficient and P-value derived from Fisher's transformation is presented. Number of mice: 12.

Fig. 5

Two quantitative trait loci (QTLs) on chromosome 1 linked to increase of conventional DCs (cDCs) in peripheral blood, and one QTL on chromosome 17 linked to that of plasmacytoid DCs (pDCs). (a) Genome-wide scan using MapManager QTX identified one QTL represented by D1MIT15 and the other represented by D1MIT387 on chromosome 1, and one QTL represented by D17MIT21 on chromosome 17. 10-based logarithm of odds (LOD) scores calculated with MapManager QTX are shown. Dotted horizontal lines indicate significant LOD threshold values determined by permutation tests. Previously described spontaneous crescentic glomerulonephritis-forming/Kinjoh (SCG/Kj)-derived QTLs (Scg-1 and Scg-2) and their 1-LOD-supported intervals are shown by bold dotted lines. The 1-LOD interval of C17 was determined by the LOD curve for Gr-1⁺ cells (see Fig. 6) and shown by bold horizontal line. (b–c) QTLs for increase of whole DCs (b), cDCs (c) and pDCs (d) in peripheral blood were inherited in a recessive manner. BSF₂ mice grouped according to genotypes of D1MIT15 (Scg-1), D1MIT387 (Scg-2) and D17MIT21 (C17) were compared using analysis of variance (anova). P-values in Fisher's protected least significant difference procedure are shown; n.s. = not significant. Numbers of mice are shown in parentheses.

Fig. 6

Quantitative trait loci (QTLs) represented by D1MIT15, D1MIT387 and D17MIT21 also influenced increase of granulocytes (D1MIT15, D1MIT387 and D17MIT21) and macrophages/monocytes (D1MIT15 and D1MIT387) in peripheral blood. (a) MapManager QTX scan to identify QTL(s) linked to increase of granulocytes and macrophages/monocytes. The pattern of the logarithm of odds (LOD) curve on chromosome 1 was similar to that of conventional dendritic cells (cDCs) (Fig. 5). The QTL on chromosome 17 for granulocyte increase was represented by D17MIT21, and its 1-LOD support interval defined C17. Spontaneous crescentic glomerulonephritis-forming/Kinjoh (SCG/Kj)-derived intervals (Scg-1 and Scg-2) and significant LOD threshold values determined by permutation tests are shown as Fig. 5. (b) QTLs for granulocytosis on chromosomes 1 and 17 were inherited in a recessive manner. (c) Mode of inheritance of QTLs for increased F4/80⁺ cells was similar to those of granulocytes and increase of cDCs. There was no QTL for monocytosis on chromosome 17. Analysis of variance (anova) was performed and P-values are shown in the same fashion as Fig. 5. Numbers of mice are shown in parentheses.

Fig. 7

Effects of Fas genotypes on leucocytosis in BSF₂ mice at 12 weeks of age. Numbers of conventional dendritic cells (cDCs) (a), plasmacytoid DCs (pDCs) (b), granulocytes (c) and macrophages/monocytes (d) in peripheral blood (PB) are shown. All these four cells are increased significantly in mice with the Fas^lpr/lpr genotype than in those with Fas^+/+ or Fas^+/lpr. Mean values ± standard error of the mean are shown are shown. P-values were determined with analysis of variance (anova) and Fisher's protected least significant difference procedure. Numbers of mice are shown in parentheses.

Fig. 8

Characterization of interactions between Fas and three non-Fas quantitative trait loci (QTLs). BSF₂ mice were classified by Fas genotypes and further classified by genotypes of any one from non-Fas QTLs. Mean values ± standard error of the mean are shown are shown.

Fig. 9

Characterization of interactions between pairs from three non-Fas quantitative trait loci (QTLs). BSF₂ mice were classified by genotypes of two QTLs from three QTLs. Mean values ± standard error of the mean are shown.