Regulation of C gamma subclass germ-line transcripts in human peripheral blood B cells.
In the present study we investigated the early steps of human IgG subclass differentiation by defining the conditions necessary for IgG subclass-specific production of germ-line transcripts in human peripheral blood B cells. Constant region of gamma-globulin (C gamma) subclass germ-line transcripts were measured using newly developed reverse transcription and polymerase chain reaction (RT-PCR) assays that were shown to be C gamma subclass-specific germ-line transcripts by size of the amplification products obtained before and after digestion with appropriate endonucleases (NarI and NcoI). In initial studies we found that C gamma 1 and C gamma 2 germ-line transcripts were constitutively expressed in total peripheral blood B cells, but not in high density sIgM+(sIgG-) B cells prepared with Percoll density gradients and magnetic beads separation techniques; the latter cells were therefore used throughout this study. Induction of germ-line transcripts (germ-line C gamma 1 transcript) was noted in stimulated B cells (SAC plus IL-2) at 6 h; thus, the appearance of germ-line transcripts could not be attributed to preferential growth of B cells expressing germ-line transcripts. In subsequent studies we found that induction of germ-line transcripts for the various IgG subclasses fell into two patterns. Induction of C gamma 3 and C gamma 1 germ-line transcripts, transcripts of genes in the first human duplication unit, generally required a proliferative stimulus (Staphylococcus aureus Cowan I, SAC) and was brought about by SAC plus IL-4 (C gamma 3 germ-line transcripts) and SAC alone or SAC plus IL-2 (C gamma 1 germ-line transcripts); in contrast, induction of C gamma 2 and C gamma 4 germ-line transcripts, transcripts of genes in the second duplication unit, was accomplished with cytokines alone: IFN-gamma (C gamma 2 germ-line transcripts) and IL-4 (C gamma 4 germ-line transcripts), and was not augmented by addition of a proliferative stimulus. Finally, we found that IFN-gamma inhibited IL-4-induced C gamma 3 and C gamma 4 germ-line transcripts (with or without SAC). These findings establish that the various human IgG subclasses manifest distinct requirements for the regulation of early steps in isotype differentiation. In addition, they suggest that human C gamma genes exhibit patterns of regulation related to their respective duplication units.
Mucosal Immunity Section, National Institute of Allergy and Infectious Diseases, Bethesda, MD 20892.
A
A
Kitani
W
W
Strober
D001402:B-Lymphocytes / Q000378:metabolism*; D001483:Base Sequence; D002478:Cells, Cultured; D005786:Gene Expression Regulation; D005803:Genes, Immunoglobulin; D006801:Humans; D007127:Immunoglobulin Constant Regions / Q000235:genetics*; D007074:Immunoglobulin G / Q000145:classification / Q000235:genetics*; D007132:Immunoglobulin Isotypes / Q000235:genetics*; D007371:Interferon-gamma / Q000494:pharmacology; D015847:Interleukin-4 / Q000494:pharmacology; D008969:Molecular Sequence Data; D016133:Polymerase Chain Reaction; D012333:RNA, Messenger / Q000032:analysis*; D011947:Receptors, Antigen, B-Cell / Q000032:analysis
D007127:Immunoglobulin Constant Regions; D007074:Immunoglobulin G; D007132:Immunoglobulin Isotypes; D012333:RNA, Messenger; D011947:Receptors, Antigen, B-Cell; D015847:Interleukin-4; D007371:Interferon-gamma