Cryoglobulinemic glomerulonephritis in thymic stromal lymphopoietin transgenic mice

Abstract

Mixed cryoglobulins are complexes of immunoglobulins that reversibly precipitate in the cold and lead to a systemic disease in humans. Renal involvement usually manifests as a membranoproliferative glomerulonephritis with marked monocyte infiltration and, at times, intracapillary thrombi. Thymic stromal lymphopoietin (TSLP) is a recently cloned cytokine that supports differentiation and long-term growth of B cells. Here we report that TSLP overexpression in mice results in the development of mixed cryoglobulins, with renal involvement closely resembling cryoglobulinemic glomerulonephritis as it occurs in humans. One hundred twenty-three mice were sacrificed at monthly intervals, with at least five TSLP transgenic mice and five controls in each group. Blood, kidneys, spleen, liver, lung, and ear were collected and studied by routine microscopy, immunofluorescence, immunohistochemistry, and electron microscopy. TSLP transgenic animals developed polyclonal mixed cryoglobulinemia (type III) and a systemic inflammatory disease involving the kidney, spleen, liver, lung, and ears. Renal involvement was of a membranoproliferative type demonstrating thickened capillary walls with cellular interposition and double contours of the basement membrane, expansion of the mesangium because of increased matrix and accumulation of immune-deposits, subendothelial immune-deposits, focal occlusion of capillary loops, and monocyte/macrophage influx. In contrast to the severe glomerular lesions, the tubulointerstitium was not involved in the disease process. The renal lesions and the disease course were more severe in females when compared to males. We describe a mouse strain in which a B-cell-promoting cytokine leads to formation of large amounts of mixed cryoglobulins and a systemic inflammatory injury that resembles important aspects of human cryoglobulinemia. This is the first reproducible mouse model of renal involvement in mixed cryoglobulinemia, which enables detailed studies of a membranoproliferative pattern of glomerular injury.

Figure 1.

A: Sera from a wild-type mouse (left) and a TSLP transgenic mouse (right) after storage at 4°C. The serum on the right demonstrates a cryoprecipitate (arrow). B: Electrophoresis and immunofixation of the cryoprecipitate of a TSLP transgenic mouse illustrates polyclonal IgG and polyclonal IgM, consistent with mixed cryoglobulins (type III). C: Serum protein electrophoresis of a TSLP transgenic male (right) and a wild-type littermate (left). The serum from the TSLP transgenic mouse shows a prominent polyclonal γ-globulin increase (arrowhead). D: Spleen from a wild-type (left) and a TSLP transgenic mouse (right) illustrates the prominent enlargement of the spleen. E: Kidneys from a TSLP transgenic mouse of normal size. Note the impression of the superior surface of the left kidney that resulted from the massive enlargement of the spleen (right).

Figure 2.

Systemic involvement in TSLP transgenic mice. A, B, and C: Lungs from TSLP transgenic mice. A perivascular leukocytic infiltrate (A) is present in the lung of a TSLP transgenic male without significant involvement of the alveoli. B: The higher magnification of A illustrates a mixed leukocyte population. C: Lung of a TSLP transgenic female at the age of 84 days. The alveoli are filled with large macrophages, some of which contain crystalline material (arrow). D, E, and F: Livers of TSLP transgenic mice. A mixed perivenous leukocyte infiltrate is present in D and E. Further spreading of the leukocyte infiltrate into the liver parenchyma is illustrated in F. G and H: Spleen of a TSLP transgenic male at the age of 111 days (G) and a wild-type control (H) at the same magnification. I: Enlarged mediastinal lymph node from a TSLP transgenic male at the age of 119 days. J: Ear of a wild-type control. K and L: Ear of a TSLP transgenic male at the age of 142 days demonstrates a prominent leukocyte infiltrate of the cutis. M: Thymus of a wild-type control. N: Illustrates the mediastinal tissue removed from a TSLP transgenic male at the age of 119 days. Original magnifications: ×40 (H, I, M, and N); ×200 (A, D, F, J, and K); ×400 (C and L); ×1000 (B and E).

Figure 3.

Time course of the renal involvement in TSLP transgenic mice. The figure illustrates the time course of the renal lesions in monthly intervals. Columns one and three represent the age-matched wild-type controls. The specimens are from mice at the age of ∼1 month (A–D), 2 months (E–H), 3 months (I–L), 5 months (M and N), and 7 months (O and P). Note the increase of mesangial matrix, which shows a severer course in female mice (arrow).

Figure 4.

Morphological features of renal involvement in TSLP transgenic mice. A: Wild-type male at the age of 199 days (silver stain; original magnification, ×1000). B: TSLP transgenic female at the age of 71 days. The glomerular tuft shows prominent increase of agyrophil mesangial matrix (arrow; silver stain; original magnification, ×1000). C and D: Transgenic male at the age of 140 days. Deposition of PAS-positive material in peripheral capillaries and the mesangium (PAS stain; original magnification, ×1000). D and E illustrate the severity of the glomerular lesion with massive deposition of PAS-positive material (D) that is not agyrophil in the silver stain (E, original magnification, ×400). Note the normal tubulointerstitium in contrast to the severe glomerular lesion. F: Thickened glomerular capillary loop with a double contour (arrow) in a 137-day-old TSLP transgenic male (silver stain; original magnification, ×1000).

Figure 5.

Time course of renal lesions in females. A: Time course of the percentage of glomerular matrix per glomerulus. B: Time course of the mean number of glomerular cells. C: Time course of macrophage infiltration expressed as mean area of macrophages per glomerulus (μm per glomerulus).

Figure 6.

Time course of renal lesions in males. A: Time course of the percentage of glomerular matrix per glomerulus. B: Time course of the mean number of glomerular cells. C: Time course of macrophage infiltration expressed as mean area of macrophages per glomerulus (μm per glomerulus).

Figure 7.

Characterization of the immune deposits by immunofluorescence. A, D, G, and J: Renal specimens from a wild-type female stained for IgM (A), IgG (D), C3 (G), and IgA (J). The tissue shows weak and focal positivity for IgM and IgG, no glomerular C3 deposition, and positivity for IgA. B, E, H, and K: Renal specimen from a TSLP transgenic female stained for IgM (B), IgG (E), C3 (H), and IgA (K). Strong granular IgM and IgG deposits can be detected in the mesangium and in capillary walls. Focal C3 deposits can be seen in the same distribution. C, F, I, and L: Renal specimen from a TSLP transgenic male stained for IgM (C), IgG (F), C3 (I), and IgA (L).

Figure 8.

Ultrastructual features of TSLP transgenic females. A: Transmission electron microscopy of a specimen from a 1-month-old TSLP transgenic female. The glomerular capillary shows a large subendothelial electron-dense deposit (asterisk), and cellular interposition (C). Note effacement of the foot processes over the deposit (V, visceral epithelial cell; E, endothelial cell). B and C: Transmission electron microscopy of a renal specimen from a 2-month-old TSLP transgenic female. Widening of the mesangial area because of increased matrix, and electron-dense deposits (D, deposit), with a tubular ultrastructure at higher magnification (arrow in C) (V, visceral epithelial cell). Original magnifications: ×4400 (A and B); ×16,900 (C).

Figure 9.

Microtubular ultrastructure of immune deposits in a TSLP transgenic male. Transmission electron microscopy of a specimen from a 199-day-old TSLP transgenic male (original magnification, ×7100). Note the prominent microtubular organization of the large mesangial immune deposit. The deposits demonstrate annular appearance on transverse sections (arrow) and a cylindrical on longitudinal sections (arrowhead).