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Identification of an IL-7-associated pre-pro-B cell growth-stimulating factor (PPBSF). II. PPBSF is a covalently
1Department of Pathology, School of Medicine, University of Connecticut Health Center, Farmington 06030, USA.
Abstract:
Evidence is provided in a companion paper for an IL-7-associated molecular complex that selectively stimulates the proliferation and presumptive differentiation of pre-pro-B cells in our long-term bone marrow culture system and "primes" them to proliferate in response to monomeric IL-7. Here, Western immunoblot analysis reveals that this pre-pro-B cell growth-stimulating factor (PPBSF) is a self-assembling heterodimer of IL-7 and a cofactor with a M(r) of 30,000. Thus, when developed with anti-IL-7 mAb, PPBSF migrates electrophoretically as a covalently bound approximately 55-kDa molecule under nonreducing conditions but dissociates under reducing conditions. Furthermore, the addition of rIL-7 or native IL-7 to medium conditioned by stromal cells from IL-7 gene-deleted (-/-) mice results in the formation of active 45-kDa and 55-kDa molecular complexes, respectively. Antiserum prepared in IL-7(-/-) mice against affinity-purified PPBSF contained separable reactivities for IL-7 and the non-IL-7 component of PPBSF. The PPBSF cofactor detected by this antiserum migrates as an approximately 30-kDa molecule and is able to maintain the viability, but not the proliferation, of pre-pro-B cells. Furthermore, the cofactor is produced constitutively by IL-7(-/-) and IL-7(+/+) bone marrow stromal cells under pro-B- but not pre-B-type culture conditions. Consequently, IL-7 appears to exist almost entirely as a heterodimer (i.e., PPBSF) in pro-B-type cultures, whereas it exists almost entirely as a monomer in pre-B-type cultures. Although the identity of the PPBSF cofactor remains to be determined, it does not appear to be stem cell factor, insulin-like growth factor-1, thymic stromal-derived lymphopoietin, flt3, stromal cell-derived factor-1, or IL-7R.
Insights
Interleukin-7 (IL-7) forms a heterodimer complex (PPBSF) with a cofactor, stimulating pre-B cell growth. This complex, crucial for early B cell development, exists as a monomer in later stages.
Area of Science:
- Immunology
- Cell Biology
- Molecular Biology
Background:
- Interleukin-7 (IL-7) is vital for lymphocyte development.
- A previously identified IL-7-associated complex stimulates pre-B cell proliferation and differentiation.
- Understanding the molecular nature of this complex is crucial for comprehending early B cell development.
Purpose of the Study:
- To elucidate the molecular composition of the IL-7-associated pre-B cell growth-stimulating factor (PPBSF).
- To characterize the role of the PPBSF cofactor in B cell development.
- To determine the conditions under which IL-7 exists as a monomer versus a heterodimer.
Main Methods:
- Western immunoblot analysis using anti-IL-7 monoclonal antibodies (mAbs) and antiserum against affinity-purified PPBSF.
- Electrophoretic analysis under nonreducing and reducing conditions.
- Culture of bone marrow stromal cells from IL-7 gene-deleted (-/-) and wild-type (+/+) mice.
Main Results:
- PPBSF is a heterodimer of IL-7 and a ~30 kDa cofactor.
- PPBSF migrates as a ~55 kDa molecule under nonreducing conditions and dissociates upon reduction.
- The PPBSF cofactor maintains pre-B cell viability but not proliferation and is constitutively produced by stromal cells under specific culture conditions.
- IL-7 exists predominantly as a heterodimer (PPBSF) in pro-B cell cultures and as a monomer in pre-B cell cultures.
Conclusions:
- IL-7 functions as a heterodimer (PPBSF) with a ~30 kDa cofactor during early B cell development (pro-B stage).
- The PPBSF cofactor is essential for pre-B cell viability and is produced by bone marrow stromal cells.
- The shift from heterodimer to monomeric IL-7 may regulate distinct phases of B cell development.