Differences in expression of toll-like receptors and their reactivities in dendritic cells in BALB/c and C57BL/6 mice - PubMed (original) (raw)

Differences in expression of toll-like receptors and their reactivities in dendritic cells in BALB/c and C57BL/6 mice

Tie Liu et al. Infect Immun. 2002 Dec.

Abstract

We have previously reported that differences in early production of interleukin 12 (IL-12) by dendritic cells (DC) underlies the difference between the susceptibilities to Listeria monocytogenes of C57BL/6 and BALB/c mice. To elucidate mechanisms for the different abilities of DC to produce cytokine in C57BL/6 and BALB/c mice, we examined Toll-like receptor (TLR) expression by DC and their responses in vitro to known microbial ligands for TLRs. We found that DC isolated from the spleens of naive C57BL/6 mice preferentially expressed TLR9 mRNA, whereas DC from naive BALB/c mice strongly expressed TLR2, -4, -5, and -6 mRNAs. C57BL/6 DC produced a higher level of IL-12p40 in response to the ligands for TLR4 (lipopolysaccharide), TLR2 (lipoprotein), and TLR9 (CpG), whereas BALB/c DC responded to these ligands by producing a larger amount of monocyte chemoattractant protein 1. C57BL/6 DC expressed higher levels of CD40 and Stat4 than BALB/c DC did, suggesting that naive C57BL/6 mice contained more-mature subsets of DC than naive BALB/c mice. Differences in reactivities of DC to microbial molecules through TLRs may be associated with susceptibility and resistance to Listeria infection in BALB/c and C57BL/6 mice.

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Figures

FIG. 1.

FIG. 1.

Comparison of maturation levels in DC from C57BL/6 and BALB/c mice. (A) Fluorescence-activated cell sorter analysis of expression of CD11c, CD40, CD86, CD8α, or MHC class II on DC. DC from naive C57BL/6 and BALB/c mice were stained with FITC-conjugated CD86 MAb, PE-conjugated CD40 MAb, and biotin-conjugated anti-CD11c MAb followed by incubation with streptavidin-Red670-Cy-chrome and were analyzed by FACScaliber. Negative controls (thin lines) were obtained by staining with control Ab. The representative profile from each strain is shown as a single histogram. (B) Expression of Stat4 in DC from naive C57BL/6 and BALB/c mice. Cell extracts from pooled DC from five mice of each strain were subjected to Western blot analysis with anti-Stat4 Abs as described in Materials and Methods. Three independent experiments showed similar results, and the results of a representative experiment are shown. (C) Detection of cytokine mRNA by RT-PCR in DC from C57BL/6 and BALB/c mice. cDNAs were amplified using primers specific for IL-12p40, MCP-1, or β-actin and were separated on a 3% agarose gel containing ethidium bromide. Three independent experiments showed similar results, and the results of a representative experiment are shown. (D) Detection of IL-12p40 and MCP-1 production by DC from C57BL/6 and BALB/c mice. DC were cultured in medium overnight, and the concentrations of IL-12p40 and MCP-1 in the supernatants were measured by ELISA. Four independent experiments showed similar results, and the data are expressed as the mean plus standard deviation of triplicate cultures of each strain in a representative experiment. *, significantly different from the values for BALB/c mice (P < 0.01).

FIG. 2.

FIG. 2.

Detection of TLR mRNA in DC from naive C57BL/6 and BALB/c mice. (A) RT-PCR analysis for TLR mRNA. Total RNAs were extracted from DC of each strain and reverse transcribed. cDNAs were amplified using primers specific for each TLR and separated on a 3% agarose gel containing ethidium bromide. (B) Northern blot analysis for TLR mRNA. Total RNAs from DC were resolved by formaldehyde gel electrophoresis, transferred to a nitrocellulose membrane, and hybridized with a mouse TLR2 or a mouse TLR4 cDNA probe. A picture of the ethidium bromide (EtBr)-stained gel is also shown. Three independent experiments showed similar results, and the results of a representative experiment are shown.

FIG. 3.

FIG. 3.

. Cytokine production by DC from C57BL/6 and BALB/c mice. DC (106/ml) pooled from five mice of each strain were cultured with various doses of LPS, lipoprotein, zymosan, or CpG-oligodeoxynucleotides (ODN) for 24 h, and the concentration of IL-12p40, MCP-1, or TNF-α in the supernatants was measured by ELISA. Four independent experiments showed similar results, and the data are expressed as the mean plus standard deviation of triplicate cultures of each strain in a representative experiment. * and **, significantly different from the values for BALB/c mice (*, P < 0.05; **, P < 0.01).

FIG. 4.

FIG. 4.

Detection of TLR2 mRNA, TLR4 mRNA, and MCP-1 production by DC from C57BL/6 mice and BALB/c mice infected with L. monocytogenes. (A) Total RNAs extracted from splenic DC from mice at indicated times after an intravenous inoculation with 6 × 104 L. monocytogenes cells. The cDNA was amplified using primers specific for TLR2 or TLR4 and separated on a 3% agarose gel containing ethidium bromide. Three independent experiments showed similar results, and the results of a representative experiment are shown. (B) Concentration of MCP-1 in 24-h culture supernatant of DC were determined by ELISA. Three independent experiments showed similar results, and the results of a representative experiment are shown. The data are expressed as the mean plus standard deviation from triplicate cultures of each strain in a representative experiment. *, significantly different from the values for BALB/c mice (P < 0.01).

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