The pathogenic role of macrophage migration inhibitory factor in immunologically induced kidney disease in the rat

Abstract

Macrophage migration inhibitory factor (MIF) plays a pivotal role in the inflammatory response in endotoxemia and in the delayed-type hypersensitivity response, but its potential as a regulator of immunologically induced disease is unknown. We have addressed this issue by administering a neutralizing anti-MIF antibody in a rat model of immunologically induced crescentic anti-glomerular basement membrane (GBM) glomerulonephritis. Six individual experiments using paired inbred littermates were performed. Rats were primed with rabbit immunoglobulin on day -5 and then injection with rabbit anti-rat GBM serum on day 0. Pairs of animals were treated with anti-MIF or a control monoclonal antibody from the time of anti-GBM serum administration until being killed 14 d later. Control antibody-treated animals developed severe proteinuria and renal function impairment with severe histological damage due to marked leukocytic infiltration and activation within the kidney. In contrast, anti-MIF treatment substantially reduced proteinuria, prevented the loss of renal function, significantly reduced histological damage including glomerular crescent formation, and substantially inhibited renal leukocytic infiltration and activation (all P <0.001 compared with control treatment). Inhibition of renal disease by anti-MIF treatment was attributed to preventing the marked upregulation of interleukin-1beta, leukocyte adhesion molecules including intercellular adhesion molecule-1 and vascular cell adhesion molecule-1, and inducible nitric oxide synthase expression seen in the control antibody-treated animals. This inhibition of progressive renal injury was mirrored by the complete suppression of the skin delayed-type hypersensitivity response to the challenge antigen (rabbit IgG). Interestingly, anti-MIF treatment did not effect the secondary antibody response or immune deposition within the kidney, indicating that MIF participates in cellular-based immunity in this primed macrophage-dependent anti-GBM glomerulonephritis. In conclusion, this study has demonstrated a key regulatory role for MIF in the pathogenesis of immunologically induced kidney disease. These results argue that blocking MIF activity may be of benefit in the treatment of human rapidly progressive glomerulonephritis, and suggest that MIF may be important in immune-mediated disease generally.

Figure 1

Effect of anti-MIF treatment on renal function in rat antiGBM disease. The effect of anti-MIF (open circles; dotted line) and irrelevant control (closed circles; solid line) antibody treatment on the following: (a) urinary protein excretion, (b) serum urea levels, (c) serum creatinine levels, and (d) creatinine clearance, was assessed at different times after injection of anti-GBM serum. Data is as the mean ± SEM for six animals. **P <0.01, ***P <0.001 versus time-matched control antibody-treated animals, and; ^a P <0.05, ^b P <0.01, ^c P <0.001 versus normal rats (day 0) by an unpaired two-sided Student's t-test.

Figure 2

Effect of anti-MIF antibody treatment on histological damage in rat anti-GBM disease. The effect of anti-MIF (gray bars) and irrelevant control (closed bars) antibody treatment on glomerular hypercellularity (Hypercell), glomerular sclerosis (Sclerosis), glomerular crescents formation (Cresc), and tubulointerstitial lesions (Int.Lesions) was determined on day 14 of rat anti-GBM disease. Data is as the mean ± SEM for six animals. ***P <0.001 versus control antibody treated by an unpaired two-sided Student's t-test.

Figure 3

Effect of anti-MIF antibody treatment on leukocyte infiltration and adhesion molecule expression in rat anti-GBM disease. The effect of anti-MIF (gray bars) and irrelevant control (closed bars) antibody treatment on glomerular and tubulointerstitial leukocytic infiltration, activation, and ICAM-1 or VCAM-1 expression was analyzed on day 14 of rat anti-GBM disease using an unpaired two-sided Student's t-test or a Mann-Whitney U test for glomerular ICAM-1 and VCAM-1 expression. Data is as the mean ± SEM for six animals. Normal rats (open bars). ns, no significant difference, *P <0.05, **P <0.001, ***P <0.001.

Figure 4

Effect of anti-MIF treatment on renal MIF mRNA and protein expression in rat anti-GBM disease. Combined in situ hybridization and immunohistochemistry (a and b) and double immunostaining (c and d) illustrate the marked upregulation of glomerular and tubulointerstitial MIF mRNA (dark blue) and protein (blue) expression in association with local macrophage (brown) accumulation, severe tubulitis (stars), and tissue injury (asterisk) in control antibody-treated anti-GBM disease (a and c), In contrast, anti-MIF antibody treatment substantially reduced renal MIF mRNA and protein, as illustrated in b and d, together with inhibition of macrophage accumulation. In addition, strong MIF expression by macrophages (purple; arrow heads) seen in control antibody-treated animals (a and c) is almost completely abolished by anti-MIF treatment (b and d). The photomicrographs from the anti-MIF–treated animals represent the areas of the kidney with least inflammatory involvement. Sections were counterstained with PAS. Magnification, ×320.

Figure 5

Quantitation of MIF expression in rat anti-GBM disease. The number of MIF mRNA-positive cells (a and b) and MIF protein stained cells (c and d) were scored in normal rat kidney (open bars), and in control (closed bars) and anti-MIF (gray bars) antibody treated anti-GBM disease. Data is expressed as the mean ± SEM for six animals. *P <0.05, **P <0.01, ***P <0.001.

Figure 6

Effect of anti-MIF treatment on IL-1β and iNOS expression in rat anti-GBM disease. Double immunohistochemical staining illustrates the following: (a) marked upregulation of IL-1β protein by both intrinsic glomerular and tubulointerstitial cells (dark blue) and infiltrating ED1⁺ macrophages (purple; arrow heads) including multinucleated giant cells (asterisk) on day 14 of control antibody-treated anti-GBM disease; (b) a substantial reduction in glomerular, and particularly tubular and macrophage, IL-1β protein by anti-MIF antibody treatment; (c) a dramatic increase in both glomerular and tubular iNOS expression (brown), including iNOS expression by ED1⁺ macrophages (purple; arrows) on day 14 of control antibody-treated anti-GBM disease; and (d), a marked inhibition of iNOS expression by anti-MIF antibody treatment. The photomicrographs from the anti-MIF–treated animals represent the ares of the kidney with least inflammatory involvement. Sections were counterstained with PAS. Magnification, ×320.

Figure 7

Quantitation of IL-1β expression in rat anti-GBM disease. (a) Glomerulus, (b) tubules. Anti-MIF treatment almost completely abrogated the upregulation of IL-1β expression seen in control-treated animals. Normal rat kidney (open bars), control-treated (CTL, closed bars), and anti-MIF–treated animals (gray bars). Data is shown as the mean ± SEM for six animals. ns, no significant difference, **P <0.01.

Figure 7

Quantitation of IL-1β expression in rat anti-GBM disease. (a) Glomerulus, (b) tubules. Anti-MIF treatment almost completely abrogated the upregulation of IL-1β expression seen in control-treated animals. Normal rat kidney (open bars), control-treated (CTL, closed bars), and anti-MIF–treated animals (gray bars). Data is shown as the mean ± SEM for six animals. ns, no significant difference, **P <0.01.

Figure 8

Quantitation of iNOS expression in rat anti-GBM disease. Anti-MIF treatment causes a marked inhibition of iNOS expression by both intrinsic glomerular and interstitial cells and macrophages. (a) Glomerulus, (b) interstitium. Normal rat kidney (open bars), control-treated (closed bars) and anti-MIF–treated animals (gray bars). Data is shown as the mean ± SEM for six animals. ns, no significant difference, *P <0.05, **P <0.01, ***P <0.001.

Figure 8

Quantitation of iNOS expression in rat anti-GBM disease. Anti-MIF treatment causes a marked inhibition of iNOS expression by both intrinsic glomerular and interstitial cells and macrophages. (a) Glomerulus, (b) interstitium. Normal rat kidney (open bars), control-treated (closed bars) and anti-MIF–treated animals (gray bars). Data is shown as the mean ± SEM for six animals. ns, no significant difference, *P <0.05, **P <0.01, ***P <0.001.

Figure 9

Effect of anti-MIF antibody treatment on the humoral immune response. ELISA analysis shows that there is no different in serum levels of specific rat anti–rabbit IgG between isotype control-treated (closed circles; solid line) and anti-MIF–treated (open circles; dotted line) animals. Normal serum IgG levels (open square; dashed line). Data is shown as the mean ± SEM for six animals.

Figure 10

Effect of anti-MIF antibody treatment on the skin DTH response. (a) Skin swelling, (b) leukocytic infiltration and activation. Anti-MIF treatment (gray bars) abrogated the skin swelling and macrophage and T cell accumulation and activation (iNOS and IL-2R expression) in the skin DTH response to specific challenge rabbit IgG. Data is shown as the mean ± SEM for six animals. ***P <0.001 versus control (closed bars) antibody treated by an unpaired two-sided Student's t-test.

Figure 10

Effect of anti-MIF antibody treatment on the skin DTH response. (a) Skin swelling, (b) leukocytic infiltration and activation. Anti-MIF treatment (gray bars) abrogated the skin swelling and macrophage and T cell accumulation and activation (iNOS and IL-2R expression) in the skin DTH response to specific challenge rabbit IgG. Data is shown as the mean ± SEM for six animals. ***P <0.001 versus control (closed bars) antibody treated by an unpaired two-sided Student's t-test.