Identification of individual interferon-producing cells by in situ hybridization.

Abstract

Individual interferon (IFN)-producing cells were identified by hybridization in situ followed by autoradiography. cDNAs corresponding to murine IFN-a and murine IFN-P labeled by nick-translation to high specific activity (24 x 10 dpm/,ug) with a-35S-labeled dATP were used as probes for hybridization with IFN mRNA in mouse C-243 cells induced with Newcastle disease virus. Control experiments with non-induced cells or with non-IFN-related labeled DNA monitored the specificity of the autoradiographic signal. Under optimal conditions of IFN induction, between 15% and 40% of the cells gave a hybridization signal with a mixture of IFN-a and -P probes. Differential hybridization with either the IFNa or -13 probe or a mixture of both, at three different time intervals after induction, revealed that only a small fraction of cells had detectable amounts of IFN-a mRNA, whereas in the majority of the positive cells IFN-(3 mRNA was present.Studies on the genetics and on the cellular origin of interferon (IFN) production have suffered from the limitation that the results represented average values obtained from cultures or from cell populations in vivo, because there was no method to identify and characterize individual IFN-producing cells at the same time. To be able to study IFN production at the single cell level is of obvious importance, because it can provide answers to several questions-for example, the number of IFN-producing cells in a given culture system or the nature and the number of IFN-producing cells in vivo as a function of the inducing agent. In the present study, we have used cDNA probes of murine IFN-a and -,B to develop a method for detecting the presence of specific mRNA by in situ hybridization. This method makes it possible to distinguish individual IFN-producing cells that can, at the same time, be stained for morphological identification. MATERIALS AND METHODSLabeling of cDNA Probes. The recombinant plasmids pMIF 1204 (carrying a partial cDNA for murine IFN-a2) and pM l3-3 (carrying the complete nucleotide sequence for murine IFN-f3) have been described (1, 2). After digestion with Pst I, the plasmids were electrophoresed through 1% agarose slab gels and the inserts [820 base pairs (bp) for IFN-a and 680 bp for IFN-,B1 were recovered from the gel slices by electroelution and chromatography on DE-52 columns. The inserts were labeled to high specific activity (2-4 x 108 dpm/,ug) with a-35S-labeled dATP (>1000 Ci/mmol; 1 Ci = 37 GBq; New England Nuclear) in a nick-translation reaction essentially as described by Haase et al. (3). Briefly, a 25-,ul reaction mixture contained 400 ng of DNA template/50 mM Tris-HCl, pH 7.4/10 mM MgCl/1 mM dithiothreitol/50,ug of nuclease-free bovine serum albumin per ml (Bethesda Research Laboratories)/30 ,uM of each dCTP, dGTP, and dTTP/250 ,JCi of a-35S-labeled dATP/5 units of DNA polymerase 1/100 pg of DNase I. The mixture was incubated at 15TC for 2 or 4 hr and incorporation of labeled nucleotides was determined by trichloroacetic acid precipitation in the presen...

Differential Expression of Interferon Alpha and Beta Induced with Newcastle Disease Virus in Mouse Macrophage Cultures

“…However, when we used a DNA probe coding for a MHC class I antigen, H-2k k or a cDNA probe for /3-actin, signal distribution was much more regular. This observation is in agreement with previous studies on mouse (Zawatzky et al, 1985) and human (Enoch et al, 1986) cells and indicates that a high degree of variation in IFN mRNA content from cell to cell may be a general feature of IFN-producing cells in vitro. However, this does not necessarily imply that cells from induced cultures exhibiting only background levels of grains did not contain IFN mRNA, since in these experiments it was difficult to estimate the hybridization efficiency and accordingly the detection limit of the in situ hybridization.…”

“…Following hybridization, the slides were washed in three changes of 1 x SSC/50~ formamide at 42 °C (IFN-ct and -/3 probes) or 30 °C (H-2k k and chicken/3-actin probe) for 2 h, dehydrated with increasing concentrations of ethanol and dried overnight. Autoradiography was performed as described (Zawatzky et al, 1985). For microscopic evaluation, grains on an average of 100 cells with intact morphology were counted.…”

Activation of the human beta-interferon gene requires an interferon-inducible factor.

“…This explains why only a fraction of the HeLa-C-127 and 143 tk--C-127 heterokaryons express human f-IFN mRNA. Heterogeneity in the expression of aand ,B-IFN in response to induction has also been reported in mouse C-243 cells (49). Variation in the responses of individual cells to inducers may be a general property of the IFN system.…”

Comparing Enhancer Action in Cis and in Trans

“…In this system, single-cell analysis showed that only a small number of cells have elevated levels of endogenous b-globin mRNA, suggesting stochastic activation of the native b-globin promoter. Similarly, expression of IFN-b in response to viral infection in humans is stochastic, with only a subset of the cells of a population showing detectable transcript (Zawatzky et al 1985). A recent analysis has led to a model wherein IFN-b expression depends on interchromosomal associations between multiple unlinked loci, which occur only in a subset of cells (Apostolou and Thanos 2008).…”