Interleukin-15 production at the early stage after oral infection with Listeria monocytogenes in mice

Abstract

We previously reported that exogenous interleukin-15 (IL-15) induces proliferation and activation of intestinal intraepithelial lymphocytes (i-IEL) in naive mice. To investigate the ability of endogenous IL-15 to stimulate i-IEL in vivo, we monitored i-IEL and intestinal epithelial cells (i-EC) in mice after an oral infection with Listeria monocytogenes. Although the populations of alphabeta and gammadelta i-IEL were not significantly changed after the oral infection, the expression level of interferon-gamma (IFN-gamma) was increased both at transcriptional and protein levels, and a conversely marked decrease in interleukin-4 (IL-4) was detected in the i-IEL on day 1 after infection as compared with before infection. The T helper 1 (Th1)-biased response of i-IEL coincided with a peak response of IL-15 production in the i-EC after oral infection. These results suggested that IL-15 produced from i-EC may be at least partly involved in the stimulation of i-IEL to produce IFN-gamma after oral infection with L. monocytogenes.

Figure 1

Vγ or Vδ usages of γδ intestinal intraepithelial lymphocytes (i-IEL) after per os (p.o.) infection with 1 × 10⁹ Listeria monocytogenes. mRNA of γδ i-IEL on days 0, 1 and 3 after infection was extracted, reverse transcribed into cDNA and amplified by the polymerase chain reaction (PCR) using primers for Cγ or Cδ and various Vγ or Vδ segments, respectively. The Southern blot of γ and δ PCR products was hybridized with Cγ2 and Jδ1, respectively.

Figure 2

Expression of cytokine mRNA in γδ intestinal intraepithelial lymphocytes (i-IEL) (a) or αβ i-IEL (b) and interferon-γ (IFN-γ) or interleukin (IL)-4 production (d) of i-IEL after per os (p.o.) infection with 1 × 10⁹ Listeria monocytogenes. mRNA was extracted from i-IEL on days 1 and 3 after p.o. infection with L. monocytogenes and reverse transcription–polymerase chain reaction (RT–PCR) was carried out with cytokine-specific primers. (a) and (b) PCR products were hybridized with an internal probe specific for each cytokine. PCR products of IL-2, IFN-γ, IL-4, transforming growth factor-β₁ (TGF-β₁) and Fas-ligand (Fas-L) were: 167 bp, 213 bp, 180 bp, 257 bp and 537 bp, respectively. The typical profiles shown are representative of two independent experiments. (c) Expression of β-actin mRNA in γδ i-IEL (left panel) or αβ i-IEL (right panel). Each sample was assayed for β-actin mRNA by competitive PCR (see the Materials and methods). The target band is the upper band. For each sample, cDNA was co-amplified in a series of three reactions with a twofold dilution series of mimic concentrations. The sizes of PCR products of target and control DNAs are 348 bp and 264 bp, respectively. The expression of each cytokine was determined by PCR-assisted mRNA amplification using an adjusted concentration of cDNA. (d) Whole i-IEL (1 × 10⁵) were cultured for 72 hr with CD3ε monoclonal antibody (mAb) and the culture supernatants were collected. The concentrations of IFN-γ and IL-4 in the culture supernatants were determined by enzyme-linked immunosorbent assay (ELISA). The data are representative of three separate examinations using pooled cells from three C57BL/6 mice and are shown as the mean of triplicate determinations ±SD. Significant differences compared with the value for normal mice are shown: *P <0·001.

Figure 3

Expression of interleukin (IL)-15 mRNA in intestinal epitherlial cells (i-EC) after per os (p.o.) infection with 1 × 10⁹ Listeria monocytogenes. (a) mRNA was extracted from i-EC on the day indicated, reverse transcribed and cDNA was amplified using specific primers for IL-15 exon 7–8, IL-15 exon 4–5 or IL-7. The polymerase chain reaction (PCR) products were resolved on 1·8% agarose gels and were Southern blotted using radiolabelled, cytokine-specific internal oligonucleotide probes. PCR products of IL-15 exon 7–8, IL-15 exon 4–5 and IL-7 were 180 bp, 215 bp and 250 bp, respectively. The typical profiles shown are representative of three (IL-15 exon 7–8, IL-7) or two (IL-15 exon 4–5) independent experiments. (b) Each sample was assayed for β-actin mRNA by competitive PCR (see the Materials and methods). The target band is the upper band. For each sample, cDNA was co-amplified in a series of three reactions with a twofold dilution series of mimic concentrations. The sizes of PCR products of target and control DNAs were 348 bp and 264 bp, respectively. The expression of each cytokine was determined by PCR-assisted mRNA amplification using an adjusted concentration of cDNA. (c) Proliferative response of CTLL-2 cells stimulated with culture supernatants of i-EC after p.o. infection with 1 × 10⁹ L. monocytogenes. CTLL-2 cells were stimulated with the 24-hr culture supernatants of i-EC, on day 1 after p.o. infection, with anti-IL-15 monoclonal antibody (mAb) (10 μg/ml) or anti IL-2 mAb (10 μg/ml). The value for the proliferative response of CTLL-2 cells without culture supernatant was 864·6±62·7 counts per minute (c.p.m.). The data are representative of two separate examinations using pooled cells from three C57BL/6 mice and are shown as the mean of triplicate determinations ±SD. *P <0·001. k.c.p.m., × 10³ c.p.m.