Identification of an IL-7-associated pre-pro-B cell growth-stimulating factor (PPBSF). II. PPBSF is a covalently linked heterodimer of IL-7 and a Mr 30,000 cofactor.
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.
Lai
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L
Department of Pathology, School of Medicine, University of Connecticut Health Center, Farmington 06030, USA.
Chen
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F
McKenna
S
S
Goldschneider
I
I
D000818:Animals; D000918:Antibody Specificity; D001402:B-Lymphocytes / Q000166:cytology* / Q000378:metabolism; D001854:Bone Marrow Cells / Q000166:cytology*; D002455:Cell Division / Q000187:drug effects / Q000276:immunology; D002478:Cells, Cultured; D054377:Chemokine CXCL12; D018925:Chemokines / Q000032:analysis / Q000737:chemistry* / Q000276:immunology / Q000378:metabolism; D019743:Chemokines, CXC; D019281:Dimerization; D004591:Electrophoresis, Polyacrylamide Gel; D007106:Immune Sera / Q000032:analysis; D015851:Interleukin-7 / Q000276:immunology / Q000378:metabolism*; D008297:Male; D008565:Membrane Proteins / Q000032:analysis; D051379:Mice; D008810:Mice, Inbred C57BL; D018345:Mice, Knockout; D008970:Molecular Weight; D051381:Rats; D011917:Rats, Inbred Lew; D013234:Stem Cells / Q000166:cytology* / Q000378:metabolism; D017154:Stromal Cells / Q000166:cytology
D054377:Chemokine CXCL12; D018925:Chemokines; D019743:Chemokines, CXC; C515256:Cxcl12 protein, mouse; D007106:Immune Sera; D015851:Interleukin-7; D008565:Membrane Proteins; C084881:flt3 ligand protein