Evaluation of lymphocyte proliferation by immunohistochemistry in the local lymph node assay

Abstract

A modified version of the local lymph node assay (LLNA) is presented, using bromodeoxyuridine to label proliferating lymphocytes. Cell counting is done on mid-sagittal sections of individual nodes under light microscopy. Two irritants (sodium lauryl sulfate and salicylic acid), four allergens of various sensitizing potential (potassium dichromate, 4-chloroaniline, neomycin sulfate and nickel sulfate) and one chemical of unknown sensitizing potential (ethyl 3-aminobenzoate) were tested either in the short protocol using three daily topical applications and/or in the long protocol with a pre-exposure step under an occluded patch. A weak T cell proliferation was noted with both irritants in the short protocol, but not in the long protocol. Potassium dichromate induced a strong proliferative response in the short protocol. A lesser sensitizing potential was detected for 4-chloroaniline, ethyl 3-aminobenzoate and neomycin sulfate but only in the long protocol. Nickel sulfate was negative in both protocols. The long protocol was the most valuable for weak or moderate sensitizers. Histological examination of nodes ruled out intercurrent processes. The present procedure offers several advantages. The use of a non-isotopic marker enables this test to be run in a routine safety assessment department and allows the preparation of permanent slides. An increased specificity is obtained by restricting cell counting to the paracortex. Moreover, the collection of individual data permits statistical analysis of the results. This method is sensitive and reproducible and may be viewed as a useful adjunct to the LLNA.

Evaluation of auricular lymph node cell lymphocyte proliferation and cytokine production as non-radioactive endpoints during murine contact allergy

“…Flow cytometric analysis and immunohistochemical assays have been developed to count BrdU incorporation into lymph node cells (Boussiquet-Leroux et al, 1995;Lee et al, 2002, Suda et al, 2002. Takeyoshi et al (2001) investigated BrdU incorporation into lymph node cells by using a cell proliferation ELISA kit but their results for a number of allergens suggested an insufficient sensitivity because the increase in lymph node cell counts was not taken into account when calculating SI values (Takeyoshi et al, 2006).…”

Safety Assessment of Salicylic Acid, Butyloctyl Salicylate, Calcium Salicylate, C12–15 Alkyl Salicylate, Capryloyl Salicylic Acid, Hexyldodecyl Salicylate, Isocetyl Salicylate, Isodecyl Salicylate, Magnesium Salicylate, MEA-Salicylate, Ethylhexyl Salicylate, Potassium Salicylate, Methyl Salicylate, Myristyl Salicylate, Sodium Salicylate, TEA-Salicylate, and Tridecyl Salicylate

“…(This was obtained with 20% Salicylic Acid.) Boussiquet-Leroux et al (1995) reported results of an LLNA performed using 5% to 20% Salicylic Acid dissolved 4:1 in acetone-olive oil (AOO). Groups of four female CD1 mice were dosed on the dorsum of the external ears with 25 µl of the test solution or the vehicle once daily on days 1 to 3.…”

Pathway analysis of gene expression in local lymph nodes draining skin exposed to three different sensitizers

“…To assess skin sensitization, murine local lymph node assays (LLNAs), which require relatively few animals in consideration of animal welfare, have been extensively used (Kimber and Weisenberger, ; OECD, ; Klink and Meade, ; Vandebriel et al , ). Although the standard LLNA endpoint is determination of the degree of 3 H‐methyl thymidine incorporation into lymphocytes (Boussiquet‐Leroux et al , ; Homey et al , ; Takeyoshi et al , ; Suda et al , ), alternative methods and endpoints have been used to reduce the number of animals or exclude the use of radioactivity. To this end, other clinical data, such as ear thickness, lymph node (LN) weight and cell counts, have been used as toxicological endpoints for assessment of chemical sensitizing potential (Homey et al , ; Vohr et al , ; Ehling et al , 2005a, b; Ku et al , ).…”