Limiting dilution analysis of TNF producing cells in C3H/HeJ mice

J Immunol. 1988 Nov 15;141(10):3422-8.

Abstract

A limiting dilution assay (LDA) that measures the frequency of TNF producing cells is described. LDA determination is based on the inhibition of growth of a highly TNF sensitive subline from the WEHI-164 fibrosarcoma by using a micro assay sensitive to single picogram amounts of recombinant murine TNF. Using such LDA, it was determined that the reported deficiency in LPS-induced TNF production in C3H/HeJ mice is a function of reduced frequency of TNF producing cells rather than a complete lack of responsiveness. In bulk culture, LPS-triggered TNF was produced by Thy-1.2 negative spleen cells with activity recovered in both G10 Sephadex adherent and nonadherent subpopulations. LPS stimulation of spleen cells from C3H/HeJ mice resulted in TNF mRNA expression as shown in both Northern blots and in situ hybridization. The frequency of TNF mRNA bearing cells in control of C3H/HeSnJ mice by in situ hybridization correlated with that found for TNF producing cells in LDA. In C3H/HeJ spleen, significantly higher numbers of TNF mRNA positive cells were found than were shown to produce TNF in LDA.

Publication types

  • Research Support, U.S. Gov't, P.H.S.

MeSH terms

  • Animals
  • Antigens, Surface
  • Cell Line
  • Cytotoxicity Tests, Immunologic* / methods
  • Female
  • Fibrosarcoma / metabolism
  • Leukocyte Count / drug effects
  • Lipopolysaccharides
  • Mice
  • Mice, Inbred BALB C
  • Mice, Inbred C3H
  • Phenotype
  • RNA, Messenger / metabolism
  • Spleen / cytology
  • Spleen / metabolism*
  • Thy-1 Antigens
  • Tumor Necrosis Factor-alpha / biosynthesis*
  • Tumor Necrosis Factor-alpha / physiology

Substances

  • Antigens, Surface
  • Lipopolysaccharides
  • RNA, Messenger
  • Thy-1 Antigens
  • Tumor Necrosis Factor-alpha