JCI - A chemokine-to-cytokine-to-chemokine cascade critical in antiviral defense

MIP-1α–dependent changes in compartmental proportions and numbers of NK cells. To evaluate and quantitate NK cells in particular compartments, spleen, blood, and liver samples were isolated from uninfected or MCMV-infected mice at a peak time of inflammation (i.e., 48 hours after infection). Normal C57BL/6-MIP-1α^+/+ (MIP-1α⁺) and C57BL/6-MIP-1α^–/– (MIP-1α^–) mice were examined. Leukocytes were prepared, and proportions and numbers of NK cells (i.e., NK1.1⁺CD3^– cells in spleen and blood and NK1.1⁺TCR-β^– cells in liver) were determined using 2-color flow cytometric and cell yield analyses. NK cell frequencies and absolute numbers were equivalent in spleen (data not shown), blood (Figure 1, a and b), and liver (Figure 1, c and d) leukocytes isolated from uninfected MIP-1α⁺ and MIP-1α^– mice. At 48 hours after MCMV infection, they declined in a similar manner in spleens of MIP-1α⁺ and MIP-1α^– mice (data not shown). In contrast, at 48 hours, MCMV-infected MIP-1α⁺ and MIP-1α^– mice had 3- and 7-fold increases in frequencies of blood NK cells (Figure 1a), respectively. Because total blood leukocyte cell yields for MIP-1α⁺ and MIP-1α^– mice were 6.5 × 10⁶ ± 5 × 10⁵/mL and 5 × 10⁶ ± 7 × 10⁵/mL, respectively, the MIP-1α^– mice had statistically significant 2-fold higher numbers of NK cells in blood (Figure 1b). Thus, infection in the presence or absence of MIP-1α induces an increase in circulating NK cells, but MIP-1α deficiency results in further accumulation of the population in blood.

Results for the liver were different from those for either spleen or blood. As total liver leukocyte numbers increased to 5 × 10⁶ ± 10⁶ from the uninfected values of 4 × 10⁵ ± 0, MIP-1α⁺ mice had 14-fold increases in NK cell numbers at 48 hours after MCMV infection (Figure 1d). In contrast, infection-induced changes in NK cell proportions were undetectable (Figure 1c), and liver leukocyte yields only increased from uninfected values of 5 × 10⁵ ± 4 × 10⁴ to 2 × 10⁶ ± 7 × 10⁵ at 48 hours after MCMV infection of MIP-1α^– mice. As a consequence, there was less than a 4-fold increase in liver NK cell numbers after infection of MIP-1α^– mice (Figure 1d). Taken together, the data demonstrate a prominent role for MIP-1α in NK cell accumulation in liver and blood in the absence of MIP-1α during infection.

Compartmentalization of IFN-γ responses. During MCMV infections, IFN-γ production is induced in NK cells, and serum and spleen leukocyte production responses peak early but subside rapidly (6, 9). To extend characterization to liver and to examine MIP-1α effects on production, ELISA assays measuring IFN-γ protein levels were performed on serum samples and spleen and liver homogenates; the homogenates were prepared from MIP-1α⁺ and MIP-1α^– mice uninfected or infected with MCMV for 24, 36, and 48 hours. Peak infection-induced serum values in both MIP-1α⁺ and MIP-1α^– mice were detected at 36 hours after infection, reaching 4 ± 0.2 ng/mL in both groups (Figure 2a). IFN-γ production in spleen homogenates was also induced at 36 hours after MCMV infection, with values of 142 ± 10 ng/mL from MIP-1α⁺ and 180 ± 15 ng/mL from MIP-1α^– mice (Figure 2b). Responses with samples from both compartments in both groups subsided by 48 hours after infection (Figure 2, a and b). IFN-γ levels in liver homogenates were strikingly different in 2 ways. First, the liver response in MIP-1α⁺ mice was sustained at 48 hours after infection, and, second, IFN-γ produced in infected MIP-1α^– livers was profoundly reduced as compared with infected MIP-1α⁺ livers (Figure 2c). At 36 hours after infection, liver IFN-γ levels were 2 ± 0.1 ng/mL in MIP-1α⁺, but were only 0.9 ± 0.04 ng/mL in MIP-1α^– mice (Figure 2c). As compared with MIP-1α⁺, MIP-1α^– mice had more than 2- to 5-fold reductions in liver IFN-γ production over time after MCMV infection. Hence, the virus-induced NK cell IFN-γ responses in serum and spleen are MIP-1α independent, but MIP-1α is absolutely required to enhance and sustain infection-induced IFN-γ production in liver.

MIP-1α deficiency and resistance to infection. Studies were carried out to characterize contributions of MIP-1α and the accompanying systemic (without localized IFN-γ) responses for establishment of antiviral states. Spleen and liver samples were isolated from MIP-1α⁺ and MIP-1α^– mice uninfected or infected with 5 × 10⁴ PFU MCMV for up to 7 days. Plaque assays demonstrated that both MIP-1α⁺ and MIP-1α^– mice had similar viral titers in spleen and liver on day 2 after infection (Figure 3, a and b). After day 4, viral replication in both spleen and liver declined dramatically in MIP-1α⁺, but remained elevated in MIP-1α^–, mice (Figure 3, a and b). Moreover, all of the MIP-1α^–, but none of the MIP-1α⁺ mice succumbed to the infection by day 7. At a higher dose (10⁵ PFU MCMV), all of the MIP-1α⁺ mice survived, and all of the MIP-1α^– mice died by day 5 (Figure 3c). Thus, absence of MIP-1α profoundly increased susceptibility to MCMV. As the MIP-1α^– mice had systemic and spleen IFN-γ responses, sensitivities resulting from the total lack of IFN-γ (i.e., IFN-γ^– mice) and a double deficiency resulting from the lack of both IFN-γ and MIP-1α (i.e. MIP-1α^–IFN-γ^– mice) were compared with that resulting from the single MIP-1α deficiency. Viral burdens in the tissue compartments were similar throughout MCMV infection in all 3 groups of deficient mice (Figure 3, a and b). Moreover, they all succumbed to lethal effects of infection with 10⁵ PFU MCMV on day 5 (Figure 3c). All mice survived infection with low dose (10⁴ PFU) MCMV (data not shown). These results demonstrate that MIP-1α^– is critical and as important as IFN-γ for survival under these conditions. Furthermore, the results suggest that local IFN-γ production is essential, but systemic IFN-γ responses are insufficient for protection against infection.

Induction of Mig expression in liver. Mig expression was evaluated to assess downstream effects of MIP-1α, NK cells, and local versus systemic IFN-γ. Total RNA was prepared from livers of uninfected or MCMV-infected MIP-1α⁺ and MIP-1α^– mice for 36, 48, or 72 hours. Liver RNA from IFN-γ^– mice infected for 48 hours was used to document the IFN-γ requirement. To evaluate NK cell contributions to liver IFN-γ and Mig expression, RNA also was prepared from uninfected or infected immunocompetent C57BL/6 and T- and B-cell–deficient C57BL/6-SCID mice; these mice had been control antibody treated or NK cell depleted by treatments with either anti-NK1.1 or anti-AGM1 antibodies. Northern blot analyses revealed a more than 200-fold induction of Mig relative to GAPDH mRNA expression at 36 hours after infection in both MIP-1α⁺ and MIP-1α^– mice (Figure 4a). In contrast, at 48 hours, Mig gene expression was sustained in MIP-1α⁺, but was below the level of detection in MIP-1α^– mice (Figure 4a).

To increase detection sensitivity and correlate IFN-γ with Mig expression, relative RT-PCR was used for quantitation. At 36 hours after infection, IFN-γ and Mig mRNA levels were induced 70- to 93-fold relative to uninfected levels in both MIP-1α⁺ and MIP-1α^– livers (Figure 4b; Table 1). At 48 hours, however, IFN-γ and Mig message levels dramatically declined in MIP-1α^– livers to less than 3-fold induction levels; however they continued to be elevated by 87- to 100-fold in MIP-1α⁺ livers (Figure 4b; Table 1). MIP-1α was not absolutely required for elevated IFN-γ or Mig mRNA, because these were detected at 36 hours after MCMV infection (Figure 4b) and were induced equally after in vivo treatments with antibody to CD3 (Figure 4c) in both MIP-1α⁺ and MIP-1α^– mice.

Increased induction intensities of IFN-γ and Mig expression were observed in control-treated C57BL/6 and C57BL/6 SCID mice at both 36 and 48 hours after infection (Figure 4d; Table 1). After NK cell depletions, however, there were greater than 5-fold reductions in infection-induced IFN-γ and Mig mRNA increases in both types of mice (Figure 4d; Table 1). Thus, MIP-1α is needed for sustained (not short-term) induction of IFN-γ and Mig mRNA in MCMV-infected livers. NK cells also are required for peak levels of expression of these genes in this compartment; and classical NK cells are major contributors to the responses.

Mig protein expression was examined by immunohistochemistry. Sections were prepared from livers of the different mice. The results showed comparable low-level induction of Mig protein predominantly in both MIP-1α⁺ and MIP-1α^– mice at 36 hours after MCMV infection (Figure 5, a and b; Table 2). At 48 hours after MCMV infection, however, Mig protein expression had increased in MIP-1α⁺ mice (Figure 5, c and e; Table 2), but was absent in MIP-1α^– (Figure 5, d and f; Table 2) mice. Mig protein was predominantly observed in sinusoidal areas (Figure 5e). It also was abundant in liver sections from SCID mice, with production levels similar to those observed in immunocompetent MIP-1α⁺ mice (Figure 5h; Table 2), but it was not detected in uninfected (Figure 5g) or in MCMV-infected IFN-γ^– mice (Table 2). Dramatic reductions in Mig protein were evident in immunocompetent and SCID mice treated with anti-NK1.1 or anti-AGM1 (Table 2). These results demonstrate the requirements for MIP-1α, NK cells, and IFN-γ in inducing sustained production of Mig protein in liver.

Role for Mig in antiviral defense. To assess the Mig contribution to antiviral defense, spleen and liver samples were isolated from MCMV-infected C57BL/6 mice that were untreated, treated with control antiserum, or treated with antiserum-neutralizing Mig. Mice were infected with 5 × 10⁴ PFU MCMV for 4 or 5 days, and viral burdens were determined. As compared with untreated or control serum-treated and infected mice, viral titers in both compartments were increased by 1 log on day 4 and 2 logs on day 5 after infection as a result of Mig neutralization (Table 3). Moreover, at a high-dose MCMV infection (10⁵ PFU), all of the mice having Mig function neutralized, but none of the untreated or control serum-treated mice, succumbed to the lethal effects and died at day 5 after infection (Table 3). These results demonstrate that Mig is a significant contributor to protection against MCMV infection.