Appearance of B220low autoantibody-producing B-1 cells at neonatal and older stages in mice

Abstract

In this study, normal adult mice carried B220(high) conventional B cells in the spleen and liver, but carried both B220(high) and B220(low) in the bone marrow. However, at the neonatal stage, only B220(low) unconventional B cells were found in all these organs. This pattern continued up to 2 weeks after birth, and at this stage autoantibodies were detected in the sera. This phenomenon was seen in all tested young mice (1-2 weeks), irrespective of their gender. Furthermore, at older stages (more than 20 weeks), B220(low) cells reappeared in the spleen and liver, and these B220(low) cells became dominant in the bone marrow. Autoantibodies also reappeared in the sera of these older mice. Cell-sorting experiments revealed that B220(low) cells were able to produce autoantibodies upon lipopolysaccharide stimuli in vitro. These results suggest that B220(low) cells appear at both neonatal and older stages as physiological responses and eventually produce autoantibodies.

Fig. 1

Age-dependent variation of lymphocytes and their subsets. (a) Number of lymphocytes; (b) identification of B220^low and B220^high cells. Lymphocytes were isolated from the liver, spleen and bone marrow at various ages (n = 4 at each point). Two-colour staining for CD3 and B220 was conducted at the indicated points of age. Numbers in the figure represent the percentages of fluorescence-positive cells.

Fig. 2

Characterization of the phenotype of B220^low and B220^high cells in very young and adult mice. Two-colour staining of B220 and other markers was conducted for the liver, spleen and bone marrow.

Fig. 3

Expression of surface immunoglobulin (Ig)M and IgD on B220^low and B220^high cells in various organs. Three-colour staining for B220, IgM and IgD was conducted. By gated analysis, the expression of IgM and IgD on B cell subsets was investigated.

Fig. 4

Autoantibodies in sera. (a) Identification by Hep-2 cells; (b) identification of anti-dsDNA antibody by enzyme-linked immunosorbent assay (ELISA). Sera were obtained from B6 mice at various ages. Diluted sera (1 : 10) were used in experiment (a) Sera of MRL-lpr/lpr mice was a positive control.

Fig. 5

Identification of autoantibodies for lymphocyte culture. (a) Anti-dsDNA antibody in culture supernatants of whole lymphocytes; (b) anti-dsDNA antibody in culture supernatants of sorted lymphocyte fractions. Lymphocytes of various sources were cultured for 3 or 7 days in the presence of lipopolysaccaride (10 µg/ml). B220^low and B220^high cell fractions were purified by fluorescence activated cell sorting. Mean ± 1 standard deviation were produced by triplicate cultures. *P < 0·05.

Fig. 6

Measurement of the level of transaminases and total bilirubin in sera at various ages. The mean value of pooled sera (n = 4) was determined.