Lupus eritematoso sistémico en ratones MRL lpr/lpm y knockouts del receptor de quimioquina CCR2

Resumen

INTRODUCTION: Systemic lupus erythematosus is an autoimmune disease with immune-complex mediated glomerulonephritis as a major manifestation and determinant of the disease outcome. The MRL/MpJ-Fas lpr/J (MRL/lpr) mouse carries a mutation in the apoptosis-related Fas gene resulting in autoreactive lymphocyte proliferation and is considered as a mouse model closely mimicking the human disease with lymphadenopathy associated with proliferation of aberrant T cells, splenomegaly, hypergammaglobulinemia with anti DNA-antibodies leading to tissue deposition and injury in various organs, including lung and kidney. The latter are mediated by immune-Complex deposition, complement activation and infiltration of inflammatory leukocytes. The renal leukocyte infiltrates consist mostly of T cells and monocytes and are major contributors to the development of progressive renal insufficiency ultimately resulting in the demise of the MRL-Fas-lpr mice. It was reported previously that steady-state mRNA levels for the chemokine (C-C motif) receptor 2 (CCR2) continuously increase in kidneys of MRL/lpr mice. Numerous studies in both human and experimental murine lupus have provided evidence that chemokines play a considerable role during the development and progression of the renal disease. Among the best studied chemokines and their respective receptors under those conditions are MCP-1 (CCL2) and its receptor CCR2 for monocytemacrophages and T cells as well as RANTES (CCL5) and its receptors CCR1 and CCR5. As CCR2 bearing mononuclear cell types appears to play a role in both the general immune response as well as the local intrarenal leukocyte infiltration in lupus mice we examined the development and progression of the disease and especially the nephritis in MRL-Fas-lpr mice by generating a CCR2 knockout mouse and backcrossing it for seven generations into the MRL-Fas-lpr background. The CCR2 receptor and CCL2 are considered to not only play a role in the local infiltration of monocyte-macrophages and T cells, but also in the general immune response as demonstrated in CCR2 and in part also in CCL2 knockout mice. METHODS: Sections of 4 µm thickness from paraffin-embedded renal tissue were routinely stained with hematoxylin/eosin (H&E), periodic acidSchiff (PAS) and silver methenamine stains. A renal pathologist blinded for the treatment performed the histopathological. Glomerular lesions were semi-quantitatively graded from 0 to 3+ for hypercellularity, mesangial matrix expansion and necrosis (0: absence, 1: mild, 2: moderate, 3: severe), as well as for the percentage of sclerotic glomeruli and presence of crescents (0: less than 10%, 1: 10-25%, 2: 25-40%, 3 more than 40%). A glomerular score was obtained from the sum of each parameter. Tubulointerstitial lesions were also graded from 0 to 3+ for peritubular and pericapillar mononuclear cell infiltrate, tubular damage, interstitial fibrosis and vasculitis. A tubulointersitial score was also defined as the sum of all parameter. Finally, perivascular lymphoproliferative mononuclear cell infiltrate was graded from 0 (absence) to 3+ (maximal intensity). CD3, ER-HR3 and Ki67-positive cells were characterised and quantified on 4-µm-thick paraffin sections from renal tissue. For quantification, positively stained cells were counted within 20 glomeruli or within 10 high power fields (630x) of tubulointerstitial tissue from 4 independent mice per group and expressed as cells per glomerulus or high power field. Direct immunofluorescence studies were performed on 5 µm, ether/ethanol-fixed, cryostat sections by using FITC-conjugated rabbit anti-mouse IgG and IgM antibody and goat anti mouse complement C3. Parafin sections (4 µm) from other tissues (lung, salivary gland, lymph node and spleen) were stained with H&E, PAS and silver methenamine stains and evaluated by light microscope. Leucocyte infiltration was determined and graded from 0 (minimal intensity) to 3+ (maximal intensity). RESULTS AND CONCLUSIONS: CCR2-deficient MRL/lpr mice developed less lymphadenopaty, had less proteinuria, had reduced lesion score, and had less infiltration by T cells and macrophages in the glomerular and tubulointersticial compartment, despite similar levels of circulating immunoglobulins and similar immune complex depositios in the glomeruli of both groups. Anti ds-DNA antibody levels, were reduced in the absence of CCR2. The frecuency of CD8+ T cells in peripheral blood was significantly lower in CCR2-deficient MRL/lpr mice. Thus CCR2 deficiency influenced not only monocyte/macrophage and T cell infiltration in the kidney but also the systemic T cell response in MRL/lpr mice. These data suggest an important role for CCR2 both in the general development of autoimmunity and in the renal involment of the lupus-like disease. These results identify CCR2 as an additional possible target for the treatment of lupus nephritis.