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

• Published in: Journal of immunological methods Vol. 337; no. 1; pp. 24 - 34
• Main Authors: Busch, Robert, Siah, Iche M., Gee, Tracy A., Hellerstein, Marc K.
• Format: Journal Article
• Language: English
• Published: Amsterdam Elsevier B.V 20-08-2008; Elsevier
• Subjects: Animals; Antigens; Biological and medical sciences; Cell Proliferation; Chemotaxis, Leukocyte; Deuterium Oxide; Dimethyl Sulfoxide - immunology; Dinitrochlorobenzene - immunology; DNA Replication - immunology; Dose-Response Relationship, Immunologic; Female; Freund's Adjuvant - immunology; Fundamental and applied biological sciences. Psychology; Fundamental immunology; Gas Chromatography-Mass Spectrometry; Heavy water labeling; Hemocyanins - immunology; Irritants - immunology; Lymph Nodes - cytology; Lymph Nodes - immunology; Lymphocyte Activation; Lymphocyte Count; Lymphocyte recruitment; Lymphocytes - immunology; Mice; Mice, Inbred BALB C; Mice, Inbred C57BL; Models, Immunological; Molecular immunology; Murine popliteal lymph node assay; Stable isotope, cell proliferation; Staining and Labeling - methods; Techniques; LN; PE(/Cy5); TCR; DMSO; IFA; Ag; KLH; PLN(A); DNCB; BM; BSA; Lymphocyte recruitment; Heavy water labeling; FITC; EM 1/2; PBS; Murine popliteal lymph node assay; CD; f; Stable isotope, cell proliferation; CFSE; EDTA; dR; M 0/1/2; MIDA; BrdU; GC/MS; Antigen; Cell proliferation; Lymphocyte recruitment;Murine popliteal lymph node assay;Heavy water labeling;Stable isotope; Animal model; Lymph node; Lymphocyte; Immunological method
• ISSN: 0022-1759; 1872-7905
• DOI: 10.1016/j.jim.2008.05.014

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 (
2H
2O) 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
2H
2O 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
2H
2O-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.