NZB serum factor (NZB-SF): B precursor cell maturation factor identified in murine lupus: I. Identification of 60-kDa glycoprotein as the major component from both spleen cell supernatant and serum

Abstract

NZB serum factor (NZB-SF), initially identified in sera of very young NZB mice, can enhance maturation and proliferation of sIg pre-B cells in marrow. In the present study, spleen cell supernatant from young NZB mice was used as a source of NZB-SF. NZB mice were treated with Corynebacterium parvum 2 weeks prior to sacrifice, and harvested spleen cells obtained at sacrifice were cultured for 24 hr in serum-free medium. One liter of spleen cell supernatant prepared in this way contained NZB-SF-like activity equivalent to that present in 10 ml of serum collected from young NZB mice. NZB-SF was purified on an affinity Chromatographie column conjugated with mouse IgG 1 monoclonal antibody (mAb) against NZB-SF. The purified NZB-SF had pI 7.8 and showed one major band of 60 kDa and a faintly stained 35-kDa band upon SDS-PAGE under nonreducing conditions. The 60-kDa NZB-SF extracted from the gel slice was also more potent in dot blot ELISA (>100 times) than the 35 kDa NZB-SF and was biologically active. After endoglycosidase F treatment, but not after treatment with a reducing agent (2-ME), the two bands merged into a single band at the 15-kDa position. Amino acid sequence analysis of endo-F treated NZB-SF indicated that the N-terminus of this protein is blocked. Serological and functional studies of affinity-purified NZB-SF have revealed that NZB-SF is distinguishable from IL-1α, IL-2, IL-3, IL-4, IL-5. IL-6, CSF-GM, IFN-γ, and TNFα. Therefore, a major component of NZB-SF(s) in the spleen cell supernatant may be an apparently novel 60-kDa glycoprotein with a single amino acid backbone. Sera and spleen cell supernatants from normal strains of mice (DBA/2, B6. or BALB/c) were also applied to the immuno-affinity column used to purify NZB-SF. It was found that trace amounts of NZB-SF are present also in serum of normal strains of mice and that spleen cells of these mice can also produce NZB-SF in vitro following stimulation with C. parvum- In SDS-PAGE, the 60-kDa NZB-SF is also the major component of NZB-SF in normal strains of mice. These results suggest that the 60-kDa NZB-SF may be of physiological importance in B cell differentiation and that this physiological factor is overproduced in autoimmune-prone NZB mice.