Heavy water labeling of DNA for measurement of cell proliferation and recruitment during primary murine lymph node responses against model antigens

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• 作者：Robert Busch ; Iche M. Siah ; Tracy A. Gee ; Marc K. Hellerstein
• 关键词：Lymphocyte recruitment ; Murine popliteal lymph node assay ; Heavy water labeling ; Stable isotope ; cell proliferation
• 刊名：Journal of Immunological Methods
• 出版年：2008
• 出版时间：20 August 2008
• 年：2008
• 卷：337
• 期：1
• 页码：24-34
• 全文大小：1013 K

文摘

Lymph node (LN) responses to antigens involve inflammatory lymphocyte recruitment and proliferation of rare antigen-specific precursors; the relative contributions of these processes have not been well quantified. The popliteal LN assay (PLNA), used for immunotoxicity screening, measures LN swelling as a surrogate of antigen-specific immunity, but nonspecific irritants cause false-positive results. Quantification of proliferating cells may improve specificity, but commonly-used biosynthetic labels (e.g., BrdU) have limitations. In vivo labeling with heavy water (²H₂O) is nontoxic and ²H incorporation into the DNA of dividing cells highly consistent, even in apoptotic microenvironments such as the thymus. Here, we have used continuous ²H₂O labeling and GC/MS analysis to quantify the cumulative fraction of recently divided cells (f) in draining LN of mice. Priming of BALB/c mice with model antigens (KLH, DNCB) increased both LN cell counts and f in responding lymphocyte subsets, whereas lymphocyte recruitment to LN by irritants (IFA, DMSO) increased cell counts but had little effect on f. Thus, antigen-driven proliferation (possibly including a bystander component) was reflected in f, whereas LN cellularity was primarily increased by recruitment. Cell counts responded differentially to changes in Ag dose and immunization with IFA, whereas f was unaffected by these variables. GC/MS analysis of ²H₂O-labeled lymphocyte DNA affords sensitive, precise measurements of fractional lymphoproliferation. Dissection of proliferation and cell recruitment by this approach may be useful for preclinical in vivo screening of novel adjuvants and immunomodulatory agents, for studying their mechanism of action, and for immunotoxicity screening in the PLNA.