Anti-Thy-1 Antibody-mediated Complement-dependent Cytotoxicity is Regulated by the Distribution of Antigen, Antibody and Membrane Complement Regulatory Proteins in Rats

Abstract

Some therapeutic antibodies as anticancer agents exert their effects through the host immune system, but the factors that predict their cytotoxicity, including complement-dependent cytotoxicity (CDC), are unclear. In the present study, we attempted to elucidate some of these factors in a preclinical model. CDC-related mesangiolysis caused by administration of the anti-Thy-1.1 antibody can be studied in the rat anti-Thy-1 glomerulonephritis model, so the model was used in this study. Three animals each were sacrificed at 0.5, 1, 8, 24 and 48 hours after i.v. administration of the anti-Thy-1.1 antibody at 1mg/kg. The distribution of the Thy-1.1 antigen and 2 membrane complement regulatory proteins (mCRPs), Crry and CD55, in three non-treated animals and the distribution of the injected antibody and C3 in the model was studied by immunohistochemistry. In the mesangial cells of the kidney, both expression of the antigen and distribution of the antibody with C3 deposition were observed with weak expression of mCRPs. There was also antigen and antibody distribution in the medullary cells of the adrenal gland and in the lymphocytes of the thymus but no C3 deposition, which was thought to be related to high expression of mCRPs. The antigen was observed in several other organs and tissues without distribution of the antibody. Cell death was only observed in the mesangial cells. These results clearly demonstrate that activation of CDC is regulated by several factors, such as distribution of the target molecule, antibody distribution and the balance among the molecules of the CDC cascade and mCRPs.

Keywords: antibody; antigen; complement-dependent cytotoxicity; membrane complement regulatory protein; rat anti-Thy-1 glomerulonephritis model.

Fig. 1.

Distribution of Thy-1.1 antigen in normal rats. A) The distribution of the Thy-1.1 antigen is shown by the intensity and frequency of positive staining. Red, orange, yellow and white indicate strong, moderate, weak and negative staining intensity, respectively. The frequency of staining is shown as the number of colored columns. Each column stands for 25%. *Other organs and tissue elements. B) Immunohistochemistry of Thy-1.1 in the kidney, adrenal gland and thymus. Positive reactions can be seen in mesangial cells of the kidney, medullary cells of the adrenal gland and lymphocytes of the cortex in the thymus. Bar = 50 µm.

Fig. 2.

Immunohistochemical and histopathological findings in the PBS- or anti-Thy-1.1 antibody-injected rats. Immunohistochemistry for the anti-Thy-1.1 antibody and C3 and HE staining are shown. Distribution of the injected anti-Thy-1.1 antibody can be seen in the kidney, adrenal gland and thymus of the antibody-treated rat but not in the PBS-treated animal. C3 deposition in the mesangial cells of the kidney can be seen in the antibody-treated animal but cannot be seen in the PBS-treated animal. There is no positive staining in the adrenal gland or thymus of the rat injected with anti-Thy-1.1 antibody. Mesangial cell death can be seen even though cell death cannot be seen in the adrenal gland and thymus of the rat injected with anti-Thy-1.1 antibody. Karyolysis in the mesangial cell (arrowheads) and infiltrations of a small number of neutrophils (arrows) in anti-Thy-1.1 antibody-injected rat are also shown. The asterisks indicate the blood vessel in the thymus. All images are from animals at 0.5 hours after injection of PBS or the antibody. Bar = 50 µm.

Fig. 3.

Changes in the mesangial cells of the kidney at 8 and 24 hours after injection of the anti-Thy-1.1 antibody in HE staining. In the animal sacrificed at 8 hours after injection, the number of mesangial cells is decreased with reduced karyolysis and increased neutrophil infiltration (arrows). In the animal sacrificed at 24 hours after injection, the mesangial area is decreased and is accompanied by capillary dilatation of the glomerulus (arrowheads). Bar = 50 µm.

Fig. 4.

Expression of mCRPs in mesangial cells and podocytes of the kidney, medullary cells of the adrenal gland and lymphocytes of the thymus in normal rat. Immunohistochemistry of Crry and CD55 is shown. Bar = 50 µm.

Fig. 5.

Categories of CDC activation in Thy-1.1 antigen-expressing cells. A) The antibody binds to the antigen, which leads to activation of the CDC pathway and then to cell death. B) The antibody binds to the antigen, but the CDC pathway is not activated because of mCRP expression. Cell death is not induced. C) The antibody does not bind to antigen, and the CDC pathway is not activated; thus, and there is no cell death.