Transcription, translation and secretion of both IgA subclasses in polyclonally activated human lymphocytes

Abstract

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.

IgG and IgA subclass mRNA-bearing plasma cells in periodontitis gingival tissue and immunoglobulin levels in the gingival crevicular fluid

“…Underlined nucleotides show where the IgA1 probe differs from IgA2 probe. The absence of cross-hybridization of the two probes was reconfirmed by the work of Islam et al [28,29]. The problem in identifying the four IgG and two IgA subclasses lies also in the high homology in their gene sequences (about 95%) [27,31].…”

“…Synthetic digoxigenin-labelled oligonucleotide anti-sense human IgG1, IgG2, IgG3 and IgG4 oligonucleotide probes were constructed (GCA CGG TGG GCA TGT GTG AGT TTT GTC ACA AGA TTT GGG CTC; TGG GCA CGG TGG GCA CTC GAC ACA ACA TTT GCG CTC AAC TGT; A AGA TTT GGG CTC TGG GCA CCG TGG GCA TGT GTG AGT TGT; AA CTC AGG TGC TGG GCA TGA TGG GGG ACC ATA, respectively) from sequences reported previously [23][24][25]28,30]. Synthetic digoxigenin-labelled oligonucleotide anti-sense human IgA1 and IgA2 gene (CC GGA TTT TGA GAG GGT GGC GGT TAG CGG GGT CTT GGA CTC GGG GTA GGC: 50 bp long) were used.…”

“…The exact mechanism by which human B cells class switch within IgG and IgA subclasses remains unclear [27]. Although extensive homology has made it difficult to analyse the immunoglobulin subclasses both at protein and mRNA level, recently the methodology for IgG and IgA mRNA subclass detection without cross-hybridization has been reported [28][29][30].…”

Cell origin of pyothorax-associated lymphoma: a lymphoma strongly associated with Epstein–Barr virus infection

“…27 During the differentiation, early B cells express IgM, while mature B cells express various subtypes of Ig as a result of class-switch recombination. Recombination to IgA in B cells is induced by transforming growth factor-b secreted from activated T cells, 28 suggesting that cytokines locally produced in the chronic pyothorax influence the class-switch recombination. In healthy individuals, IgA 1 is secreted mainly in sputum and breast milk.…”

Studies of the molecular basis of IgA production, subclass regulation and class-switch recombination in IgA nephropathy patients

“…Solution and in situ hybridization were carried out with specific RNA probes for the 1 and 2 immunoglobulin heavy chain constant region as described previously [27,28].…”