Transcription, translation and secretion of both IgA subclasses in polyclonally activated human lymphocytes.
To analyze human IgA subclass-specific mRNA we have developed RNA probes for quantitation of alpha 1 and alpha 2 heavy chain constant region genes by solution hybridization. Under our assay conditions, we can detect as little as 3 pg of specific mRNA and there is less than 2% cross-reactivity between the two IgA subclasses. Using this method, the relative proportions of IgA1 and IgA2 mRNA in pokeweed mitogen (PWM)-stimulated cells were found to be 66% and 34%, respectively, while in the Epstein-Barr virus (EBV)-stimulated cells they were 77% and 23%, respectively. Cytoplasmic staining of the plasma cells and determination of IgA subclass distribution by flow cytometry revealed an almost even distribution of the two IgA subclasses as induced by both activators. The culture supernatant contained 72% IgA1 and 28% IgA2 after PWM stimulation, while EBV stimulation induced 85% IgA1 and 15% IgA2. This report thus describes a method for the quantitative analysis of IgA subclass-specific mRNA. Furthermore, we present evidence that in response to in vitro stimulation of peripheral blood lymphocytes by polyclonal activators the IgA-producing B cells not only synthesize both isotypes but also have the potential to secrete them.
Islam
K B
KB
Center for Biotechnology, Karolinska Institute at NOVUM, Huddinge, Sweden.
Hassan
M S
MS
Engström
P E
PE
Hammarström
L
L
Smith
C I
CI
D000911:Antibodies, Monoclonal; D001483:Base Sequence; D002460:Cell Line; D005786:Gene Expression Regulation / Q000276:immunology; D004854:Herpesvirus 4, Human; D006801:Humans; D007070:Immunoglobulin A / Q000145:classification / Q000235:genetics / Q000378:metabolism*; D008213:Lymphocyte Activation / Q000276:immunology*; D008969:Molecular Sequence Data; D009693:Nucleic Acid Hybridization; D011043:Pokeweed Mitogens; D014176:Protein Biosynthesis; D011499:Protein Processing, Post-Translational; D012333:RNA, Messenger / Q000096:biosynthesis; D014158:Transcription, Genetic
D000911:Antibodies, Monoclonal; D007070:Immunoglobulin A; D011043:Pokeweed Mitogens; D012333:RNA, Messenger