Purification to homogeneity of a novel human B cell differentiation factor.

By immunization with a partially purified form of a novel human B cell differentiation factor, 446-BCDF, we generated a series of mAbs that inhibit the functional activity of this cytokine (induction of Ig secretion by Staphylococcus aureus Cowan I strain organisms-activated B cells). Two mAbs, 929 and 204, were shown to be specific for 446-BCDF in that they failed to inhibit B cell differentiation in response to other known cytokines (IL-2, IL-6) or mitogens (PWM). More importantly, passage of crude 446-BCDF over a mAb 929-Sepharose 4B column resulted in the depletion of 446-BCDF activity, which could be recovered after elution with 0.1 M glycine, pH 2.8. Finally, TCA precipitation of column-eluted fractions was run on SDS-PAGE. Two bands corresponding to the previously described m.w. range of 446-BCDF were detected in the eluate, but not in the effluent column fractions. Furthermore, protein eluted from these bands resolved with nondenaturing gels also demonstrated 446-BCDF activity. Thus, together these data further support the existence of a novel human B cell differentiation factor that has been purified to homogeneity.

B cell differentiation factor-induced B cell maturation: regulation via reduction in cAMP.

We have previously described a novel human B cell differentiation factor (BCDF), 446-BCDF, that is distinct biochemically and functionally from other cytokines. Since signal transduction pathways involved in human B cell differentiation have been incompletely studied and are poorly understood, we assessed the effects of 446-BCDF on various intracellular second messenger systems. After exposure of B cells to 446-BCDF, intracellular cAMP concentration started to decrease at 5 min and was significantly lower at 30 min and reached the lowest level at 4 hr. In most cases, cAMP concentrations returned toward baseline by 24 hr. A cAMP analog (dibutyryl cAMP), a stimulator of adenyl cyclase (forskolin), and phosphodiesterase inhibitors (aminophylline and IBMX) which inhibited the 446-BCDF-induced decrease in intracellular cAMP, inhibited 446-BCDF-induced B cell differentiation, suggesting that the fall in intracellular cAMP was a critical event in this process. To understand the mechanism involved in the reduction of cAMP, B cells were treated with pertussis toxin (PTX), a Gi protein inhibitor. Pertussis toxin blocked 446-BCDF-induced B cell differentiation as well, suggesting that 446-BCDF may function by stimulation of a Gi-linked receptor resulting in the inhibition of adenylate cyclase with a consequent reduction in cAMP. Other cytokines known to promote Ig secretion (IL2 and IL6) also caused a reduction in cAMP, suggesting that this pathway may be generally important in B cell differentiation. Taken together, these data suggest that at least one pathway of terminal maturation in B cells may involve the reduction of intracellular cAMP.

Cytokines regulating human B cell growth and differentiation.

Utilizing a model system of anti-CD3-stimulated T cells, we have identified a potent B cell differentiation factor (BCDF) in the supernatant of these cells. This factor, 446-BCDF, appears to act on SAC-activated B cells inducing a 10- to 100-fold increase in Ig secretion. 446-BCDF has an apparent MW of 32 kDa and a pI of 6.0. Its activity cannot be inhibited by an anti-IL-6 antiserum, and activity is enhanced after passage over an anti-IL-6 affinity column. 446-BCDF activity is detected in the 50 mM salt fraction eluted from a Mono Q column. This 50 mM fraction has the only activity detected after passage over an anti-IL-6 affinity column and migrates with an apparent pI of 6.0. Taken together these data suggest that 446-BCDF is a unique potent polyclonal human BCDF which may be a predominant factor regulating terminal B cell differentiation.