Role of interleukin-12 in primary influenza virus infection

Abstract

The effect of endogenous interleukin-12 (IL-12) on the influenza virus immune response in BALB/c mice was evaluated. Following primary influenza virus infection, IL-12 mRNA and protein are detected in the lung, with live virus being required for cytokine induction. Endogenous IL-12 contributes to early NK cell-dependent gamma interferon (IFN-gamma) production (days 3 and 5) but not late T-cell-dependent IFN-gamma secretion (day 7). IL-12 contributes to the inhibition of early virus replication but is not required for virus clearance. IL-12 also modestly contributes to the activation of cytotoxic T lymphocytes. Thus, in this model of experimental influenza virus infection, endogenous IL-12 contributes primarily to the early development and activation of the innate immune response.

FIG. 1

Detection of IL-12 p40 mRNA in the lungs of BALB/c mice infected with PR8 virus. Lung tissue samples from four PR8-infected mice were collected daily on days 1, 2, and 3 postinfection, and lung tissue samples from two PR8-infected mice were collected on days 4, 5, and 6 postinfection. On days 1, 2, and 3, lung tissue was collected from mock-infected mice. Each sample was simultaneously assayed for cyclophilin as an internal control for equal RNA usage. The p40 signal was corrected against the internal control. Quantitation of the p40 mRNA content in each sample was achieved by comparing the signal strength to that of a riboprobe standard curve with known amounts of RNA subjected to the same treatment as the RNA samples. Values are the means ± standard errors of samples. ∗∗∗, P < 0.005. n.s., not significant by Student’s t test. N.D., not detectable.

FIG. 2

Cytokine production in BAL fluid of BALB/c mice infected with PR8 virus. (A) IL-12 p40 was measured by RIA. Data are from 15 BAL fluid samples from PR8-infected mice and 10 BAL fluid samples from mock-infected mice (five separate experiments). (B) IL-12 p70 was measured by capture assay. Eight BAL fluid samples from PR8-infected mice and four BAL fluid samples from mock-infected mice were collected at each time point (two separate experiments). (C) TNF-α was measured by ELISA. Data are from six BAL fluid samples from PR8-infected mice and three BAL fluid samples from mock-infected mice (three separate experiments). (D) IFN-γ was measured by RIA. Data are from 15 BAL fluid samples from PR8-infected mice and 10 BAL fluid samples from mock-infected mice (five separate experiments). Values are means ± standard errors of samples from individual mice. ∗∗, P < 0.01; ∗∗∗, P < 0.005. n.s., not significant by Student’s t test of the comparison between groups treated with noninfectious allantoic fluid and with PR8 virus.

FIG. 3

Live virus is required for the production of proinflammatory cytokines. BALB/c mice were infected intranasally with 20 μl of 10⁶ HAU of purified PR8 virus (per ml) that had been exposed to UV light. BAL fluid was collected 3 days after infection. (A) IL-12 p40 was measured by RIA. (B) TNF-α was measured by ELISA. (C) IFN-γ was measured by RIA. Experiments included a total of six mice (three separate experiments). Values are means ± standard errors of samples. ∗∗∗, P < 0.005. n.s., not significant by Student’s t test of the comparison between groups treated with noninfectious allantoic fluid and with PR8 virus.

FIG. 4

Endogenous IL-12 is required for IFN-γ production in the lungs during the early phase of influenza virus infection. PR8-infected mice were injected with normal rat Ig (100 ng) or anti-IL-12 antibody (100 ng) on days −1, 2, and 5 of infection. BAL fluid was collected on days 3, 5, and 7 postinfection and assayed for the presence of IFN-γ by RIA. Experiments included a total of 45 mice (three mice per group per experiment in five separate experiments). Values are means ± standard errors of samples. ∗∗, P < 0.01. n.s., not significant by Student’s t test.

FIG. 5

Effect of NK cell depletion on IFN-γ production. One day prior to infection with PR8 virus and 4 days postinfection, a total of 18 (three separate experiments) BALB/c mice were injected i.p. with 200 μl of αAsGM1 rabbit antimouse polyclonal antibody diluted 1:10 in PBS. Eighteen control mice were injected with normal rabbit serum (nrs). BAL fluid was collected on days 3 and 7 postinfection and assayed for the presence of IFN-γ by RIA. (B) To confirm in vivo depletion of NK cells by αAsGM1, noninfected mice treated with αAsGM1 or normal rat serum were injected i.p. with 100 μg of poly(I-C) 18 h prior to spleen removal and splenocyte NK cell activity was measured by ⁵¹Cr release with the NK cell-sensitive YAC-1 cell line. Values are means ± standard errors of samples. ∗∗, P < 0.01. n.s., not significant by Student’s t test of the comparison between groups treated with normal rabbit serum and with αAsGM1 serum. E:T ratio, effector/target cell ratio.

FIG. 6

Effect of endogenous IL-12 on virus titer. Lung tissue was collected from individual PR8-infected mice treated with normal rat Ig or anti-IL-12 at 1, 2, and 5 days. Virus titer was determined as described in Materials and Methods. Experiments included three mice per time point per experiment for three separate experiments. Values are means ± standard errors of samples. ∗∗∗, P < 0.005. n.s., not significant by Student’s t test of the comparison between groups treated with normal rat Ig and with PR8 virus. TCID₅₀, 50% tissue culture infective dose.

FIG. 7

Effect of endogenous IL-12 on CTL activity. The spleens of PR8-infected mice treated with neutralizing anti-IL-12 antibody (C17.8) or control antibody (rat IgG) were collected at 10 days postinfection, and splenocytes from individual mice were cultured with irradiated splenocytes infected with PR8 virus in vitro as stimulators. After 4 days, the cultured cells were tested as effector cells against ⁵¹Cr-labeled PR8-infected P815 target cells at the indicated effector/target cell ratios in a ⁵¹Cr release cytotoxicity assay. Experiments included four mice per experimental condition (two separate experiments). Values are means ± standard errors of samples. ∗, P < 0.05; ∗∗, P < 0.1. n.s., not significant by Student’s t test of the comparison between groups treated with normal rat Ig and with PR8 virus.