The production of allergen‐specific IgE antibodies (Abs) in allergen‐sensitized patients or animals has a mutual relationship with the immunologic response leading to allergic rhinitis. We recently reported that, after an intranasal injection of cedar pollen into mice, an interleukin‐4 (IL‐4)‐dependent increase in serum nonspecific IgE Abs was a prerequisite for the production of serum allergen‐specific IgE Abs. Here, we explored which lymphoid organs were responsive to the intranasally injected allergen and how IL‐4 and IgE Abs were produced in the lymphocytes. Time‐dependent changes in the total cell numbers and in in vitro IgE Ab production in various lymphoid organs revealed that the submandibular lymph nodes were the main responsible organ. After treatment with allergen (for IgE production) or allergen and complete Freund's adjuvant (for IgG production), we separated submandibular lymph node cells into macrophage‐, lymphocyte‐, and granulocyte‐rich populations by discontinuous Percoll density‐gradient centrifugation. Unexpectedly, bulk cells, but not the lymphocyte‐ or macrophage‐rich populations, produced significant amounts of IL‐4, IgE, and IgG; whereas production was restored by addition of Mac‐1+ cells from the macrophage‐rich to the lymphocyte‐rich fraction. Furthermore, a combination of the lymphocyte‐rich population (for IgG [or IgE]) production) and the macrophage‐rich population (for IgE [or IgG]) production) produced a large amount of IgE (or IgG). These results indicate that, in the initiation of allergic rhinitis, macrophages in the submandibular lymph nodes are essential not only for IL‐4 or immunoglobulin production, but also for class switching of immunoglobulin in lymphocytes.
Extracellular vesicles (EVs) are ubiquitously secreted
by almost
every cell type and are present in all body fluids. Blood-derived
EVs can be used as a promising source for biomarker monitoring in
disease. EV proteomics is currently being analyzed in clinical specimens.
However, their EV proteomics preparation methods are limited in throughput
for human subjects. Here, we introduced a novel automated EV isolation
and sample preparation method using a magnetic particle processing
robot for automated 96-well processing of magnetic particles for EV
proteomics analysis that can be started with a low volume of multiple
clinical samples. The automation of EV purification reduced the coefficient
of variation of protein quantification from 3.5 to 2.2% compared with
manual purification, enabling the quantification of 1120 proteins
in 1 h of MS analysis. This automated proteomics EV sample preparation
is attractive for processing large cohort samples for biomarker development,
validation, and routine testing.
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