Increased survival and reduced renal injury in MRL/lpr mice treated with a human Fcγ receptor II (CD32) peptide

Abstract

Systemic lupus erythematosus (SLE) is a multisystem chronic inflammatory disease affecting many organs. The deposition in kidney tissue of immune complexes and their interaction with macrophages is thought to trigger the inflammatory response leading to glomerulonephritis. It has been demonstrated that inhibition of this interaction in murine models can alleviate the disease. Six synthetic peptides were derived from the membrane-proximal extracellular domain (EC2) of human Fcγ receptor II (huFcγRII). Of these, one peptide, huRII6, was shown to be a potent competitive inhibitor of IgG binding to recombinant FcγRII in vitro. To examine the possible therapeutic impact of huRII6 in vivo, this peptide, or a control, was given by subcutaneous injection to female MRL/lpr mice from weeks 7 to 36, resulting in an enhanced survival rate compared with control-treated animals and a reduction of proteinuria. Histopathological examination of the kidneys showed a reduction in deposition of immune complexes and preservation of structure. Such a functional peptide should prove useful for examining the role of IgG-FcγR interactions in experimental models of disease and may provide for the development of FcR-targeting drugs to treat autoimmune disorders.

Figure 1

Inhibition of IgG binding to soluble human Fcγ receptor II A (FcγRIIA). Horseradish peroxidise–IgG was pre-incubated, respectively, with the peptide huRII6 at different concentrations (0.1–70 μm) at 4° for 2 hr using control peptide and BSA as negative control. The samples were then applied to recombinant huRII-coated plates for IgG binding for 1 hr at 37°. After washing, the absorbance of the samples was measured at 450 nm. The results shown represent the means ± SEM from three independent experiments.

Figure 2

Inhibition of IgG–receptor interaction by the peptide huRII6. FITC-labelled IgG aggregates were pre-incubated with various concentrations(1–640 μm) of the huRII6 or control peptide or BSA at 4° for 2 hr, and then applied to the transfected COS 7 cells. (a) The peptide huRII6 inhibited the binding of human IgG to the transfected COS 7 cells with an IC₅₀ value of 112.3 μm. (b) The BSA, control peptide, huRII6 inhibition of IgG binding to huFcγRII on the COS-7 cell surface by FACS. (I) 640 μm BSA; (II) 640 μm control peptide; (III) 640 μm huRII6. Non-transfected COS 7 cells were used as control. All experiments were carried out in triplicate and the binding of IgG to the COS 7 cell line given by the sample without huRII6 in three experiments was taken as 100%. The inhibition percentages (IC%) are shown as means ± SEM on the curve.

Figure 3

Treatment of young MRL/lpr mice with the peptide huRII6 results in prolonged survival and delayed development of nephritis. (a, b) Progressive age-related elevation of serum anti-dsDNA (a) and anti-ssDNA (b) antibody titres. (c) Proportion of mice without severe proteinuria (≥ 3+ on at least two consecutive examinations) versus age in weeks is shown. A decreased proportion of huRII6 peptide-treated mice had severe proteinuria compared with those treated with control peptide (P < 0.05). (d) Survival was significantly prolonged in mice treated with a huRII6 peptide of determinants compared with control. At 38 weeks, survival was 80% in the huRII6 peptide group compared with 20% in control group (P < 0.05).

Figure 4

Haematoxylin and eosin staining of kidney sections from control-treated and huRII6 peptide-treated mice. Haematoxylin & eosin staining of kidney sections from (a) control mice (n = 10) at week 37 ± 3 showing typical signs of severe glomerulonephritis including tubular dilatation and proteinaceous casts. In contrast, histological morphology of age-matched (b) huRII6 peptide-treated mice (n = 10) is essentially normal (magnification 100×).

Figure 5

Immunohistochemical examination of kidneys from huRII6 peptide-treated and control MRL/lpr mice. Kidney sections of (a) control mice (n = 10) and (b) huRII6 peptide-treated mice (n = 10) at week 37 ± 3, were stained using anti-mouse IgG to examine for IgG immune complex deposition (magnification 100×). The entire group of treated and control animals was examined by immunohistochemistry with similar results.