Antigen-specific immunoglobulin E+ B cells are preferentially localized within germinal centres

Abstract

Allergen-specific immunoglobulin E (IgE) mediates immediate-type hypersensitivity reactions and plays a central role in allergic diseases. Although antigen-driven B-cell maturation and isotype switching occur within germinal centres (GCs), the role of GCs in IgE production is poorly understood. In view of this, we investigated the development of IgE-expressing cells within GCs in response to an extensively characterized antigen, 2-phenyloxazolone (phOx). The phOx-specific IgE-expressing cells localized within GCs 7 days after immunization, and peaked in number on day 11. Surprisingly, very few IgE-positive cells were found in the T-cell areas of the lymph node. Flow cytometric studies confirmed that IgE was expressed by B cells and was not the result of trapping by follicular dendritic cells. The specificity of the antibody response was confirmed by microdissection and reverse transcription-polymerase chain reaction using phOx-specific IgE primers. IgE-positive cells were primarily found within GCs while, in contrast, many IgG1-positive cells could also be detected outside GCs in the T-cell areas. Taken together, these data highlight the importance of GCs in the production of antigen-specific IgE antibody.

Figure 1

Serum IgE concentrations following immunization with phOx. Total serum IgE concentrations were determined by a sandwich enzyme immunoassay before (day 0) and 11 days after immunization. Dots represent serum IgE levels for individual mice. There was a statistically difference between groups (P < 0·001; Mann–Whitney Rank Sum test).

Figure 2

IgE and IgG1 expression within GCs in response to phOx. Brachial lymph node sections were stained with PNA to identify GCs and either anti-IgE or anti-IgG1 antibodies by immunohistochemistry. IgE⁺ cells can be observed within GCs at day 7 (a) and day 9 (b) following immunization. In contrast, IgG1⁺ cells were found both inside and outside (arrow) GCs at day 9 (c). GCs (PNA⁺ area) are outlined by a dotted line. Original magnification is 40 ×.

Figure 3

Kinetics of GC formation and IgE/IgG1 expression following immunization with phOx. Brachial lymph node frozen tissue sections were stained with PNA to identify GCs, and antibodies against either IgE or IgG1 to determine: (a) mean number of GCs per lymph node, (b) percentage of GCs containing IgE⁺ and IgG1⁺ cells, and (c) numbers of IgE⁺ and IgG1⁺ cells within each GC. Four lymph node sections were examined from each mouse and three to five mice were used at each time-point. The GC area was determined using an ocular grid. Standard deviation bars are shown.

Figure 4

Flow cytometry analysis of IgE expression on GC B cells. Brachial lymph nodes were harvested on day 11 after phOx-BSA injection and single cell suspensions were stained for flow cytometric analysis. (a) Large B cells were identified based on CD45R (B220) expression and high forward-angle light scatter (channel > 384). (b) Histogram showing IgE expression by GL7⁺ large B cells (CD45R⁺ cells with high forward-angle light scatter). For comparison, the staining profile to an irrelevant control antibody is indicated by the light grey line. (c) Dot plot of gated cells (large, CD45R⁺ B cells) showing CD23 expression on GC B cells based on GL7 (LY77) staining. Data are representative of two separate experiments.

Figure 5

GCs express Ox-specific mRNA for IgE. Cells were retrieved from individual GCs by microdissection and examined for Ox-specific IgE mRNA by nested RT-PCR. (a) A tissue section was stained with PNA after microdissection. (b) GC cells (day 7, lane 1; day 9, lane 2) and brachial lymph node cells (day 9, lanes 3 and 4) were examined for phOx-specific IgE mRNA following immunization with phOx (lanes 1, 2 and 4) or NP (lane 3). Molecular weight markers appear in the far left lane. The expected phOx-specific mRNA product is 284 base pairs (bp) in length.