JCI - Type I interferons regulate inflammatory cell trafficking and macrophage inflammatory protein 1α delivery to the liver

Liver NK cell accumulation during MCMV infection. NK cells accumulate and localize in liver inflammatory foci during MCMV infections of C57BL/6 mice (1, 23). To characterize NK cell accumulation in another strain of mice, H&E-stained liver sections were prepared from C57BL/6 and 129 mice that were uninfected or infected with MCMV for 48 hours. The focal clustering of nucleated cells between portal areas and central veins typical of NK cell inflammation was observed following infections in both strains (Figure 1, a and b). To quantitate the NK cell yields (NK1.1⁺TCR-β^– or DX5⁺TCR-β^–) in this compartment, leukocytes were prepared, and NK cell proportions and numbers were determined using flow cytometric and total cell recovery. The liver leukocyte yields increased following infection to 4 × 10⁶ ± 3 × 10⁵ from the uninfected values of 2 × 10⁶ ± 2 × 10⁵ in C57BL/6 mice, and to 3 × 10⁶ ± 4 × 10⁵ from the uninfected values of 1 × 10⁶ ± 1 × 10⁵ in 129 mice. Although liver NK cell percentages and yields were higher in C57BL/6 mice, both C57BL/6 and 129 mice demonstrated twofold increases in frequencies (Figure 1, c and e) and fourfold increases in absolute numbers (Figure 1, d and f). Thus, both strains exhibit similar liver histological characteristics and induced accumulation of NK cells during infection.

Induction of IFN-α/β protein in liver. As IFN-α/β can have profound effects on cell trafficking (13–16) and on cytokines and chemokines (11, 12), liver IFN-α/β expression was examined by immunohistochemistry. Tissue sections were prepared from uninfected or MCMV-infected C57BL/6 (Figure 2, a–c) or 129 (Figure 2, d–f) mice. IFN-α/β was not detected in uninfected mice (Figure 2, a and d). However, at 36 and 48 hours after challenge, production was dramatically induced in both strains. Positive cells were detected in sinusoids and as scattered populations within parenchyma (Figure 2, b, c, e, and f). To quantitate type 1 IFN proteins, ELISA assays measuring IFN-α were performed on liver homogenates. At 36 hours, C57BL/6 mice had IFN-α values of 66 ± 3 and 129 mice had values of 67 ± 17 ng/g liver. At 48 hours after infection, C57BL/6 mice had 44 ± 12 and 129 mice had 64 ± 5 ng/g liver. IFN-α was below the limit of detection (<7 ng/g liver) in uninfected mice. Taken together, these results demonstrate that both strains produce IFN-α/β in liver during MCMV infection.

IFN-α/β deficiency and resistance to MCMV infection. To determine the effects of IFN-α/β responses for antiviral defense, H&E-stained liver sections were prepared from mice deficient in IFN-α/β–mediated functions (IFN-α/βR^–) and infected with MCMV for 48 or 72 hours. In contrast to immunocompetent (IFN-α/βR⁺) mice (Figure 1b), there was a profound inhibition of inflammatory foci in liver from IFN-α/βR^– mice at both 48 (Figure 3a) and 72 (Figure 3b) hours after infection. Cells having the morphological characteristics of MCMV infection, i.e., cytomegalic inclusion bodies, were frequently seen at 48 hours and were readily visible, along with necrotic foci, at 72 hours after infection (Figure 3, a and b). Thus, virus-induced liver pathology was elevated in the absence of IFN-α/β–mediated functions. To further examine the importance of IFN-α/β for antiviral defense, IFN-α/βR⁺ and IFN-α/βR^– mice were infected with a lower dose (1 × 10⁴ PFU) of MCMV and monitored for survival. All of the IFN-α/βR^–, but none of the IFN-α/βR⁺, mice succumbed to infection by day 5 (Figure 3c). Therefore, under both moderate- and low-dose conditions of infection the absence of IFN-α/β–mediated functions profoundly increases susceptibility to MCMV.

IFN-α/β effects on NK cell infiltrates and MIP-1α induction in response to infection. NK cell inflammation is important in promoting defense against MCMV in livers, and our previous studies have shown the critical role of MIP-1α for this response (1, 5). To evaluate the effects of IFN-α/β on NK cell accumulation, liver leukocytes were prepared and analyzed from IFN-α/βR⁺ and IFN-α/βR^– mice that were uninfected or infected with MCMV for 48 hours. Although liver NK cell frequencies were equivalent in both IFN-α/βR⁺ and IFN-α/βR^– mice (data not shown), significant differences in NK cell numbers were evident under both moderate-dose (Figure 4a) and low-dose (Figure 4c) conditions of infection. As total liver leukocyte yields increased, from the uninfected values of 1 to 2 × 10⁶ per g liver to 3.5 × 10⁶ per g liver after moderate-dose and to 4.5 × 10⁶ per g liver after low-dose MCMV infection, IFN-α/βR⁺ mice had twofold elevations in NK cell numbers induced by challenge (Figure 4, a and c). In contrast, liver leukocyte yields from IFN-α/βR^– mice were only 1 × 10⁶ per g liver after moderate-dose and 9 × 10⁵ per g liver after low-dose infection, compared with the uninfected values of 2 × 10⁶ per g liver. As a result, there were twofold reductions in liver NK cell numbers at 48 hours MCMV infection of IFN-α/βR^– mice (Figure 4, a and c). Although there were decreases in NK cell numbers in other compartments during infections of the IFN-α/βR^– mice at the moderate dose, these were not observed during infections at the lower dose (data not shown). Therefore, the reductions in NK cells infiltrating the liver were not a consequence of generalized NK cell deficiencies. Hence, IFN-α/β functions can promote accumulation of NK cells in the liver during infection.

To determine whether IFN-α/β modified induction of MIP-1α, ELISA assays were performed with liver homogenates prepared from IFN-α/βR⁺ or IFN-α/βR^– mice that were uninfected or infected with MCMV for 48 hours. Under the conditions of both moderate-dose (Figure 4b) and low-dose (Figure 4d) infection, MIP-1α production was induced in livers of both groups of mice. The responses observed in IFN-α/βR^–, however, were dramatically reduced compared with those in infected IFN-α/βR⁺ mice (Figure 4, b and d). By comparison to IFN-α/βR⁺, IFN-α/βR^– mice had three- to fourfold reductions in the levels of liver MIP-1α protein under both conditions of moderate- and low-dose infections. Thus, IFN-α/β is necessary for initiation of MIP-1α expression in liver during MCMV infection.

Induction of MIP-1α and accumulation of NK cells following rIFN-α administration. As demonstrated above, IFN-α/β–mediated functions are necessary to effectively control virus (Figure 3) as well as to promote MIP-1α induction and NK cell accumulation during infection (Figure 4). To evaluate the immunoregulatory effects of IFN-α/β in the absence of secondary effects resulting from increased virus-induced disease, the consequences of treating uninfected mice with rIFN-α were examined. MIP-1α protein was measured in liver homogenates prepared from vehicle- or rIFN-α–treated C57BL/6 and 129 mice. The levels of MIP-1α were low (0.08 ng/g liver) in vehicle-treated C57BL/6 and 129 mice (Figure 5, a and c). In contrast, MIP-1α protein was dramatically induced to values of 0.5 ng/g liver in C57BL/6 and 0.2 ng/g liver in 129 mice after rIFN-α administration (Figure 5, a and c). Thus, rIFN-α exposure result in greater than two- to sixfold inductions of MIP-1α in livers.

To examine the contribution of rIFN-α to NK cell accumulation, liver leukocytes were prepared and analyzed. Mice receiving rIFN-α had two- to threefold increases in the proportions of NK cells when compared with mice receiving vehicle treatments (data not shown). In both C57BL/6 and 129 mice, the NK cell numbers increased from the vehicle-treated values of 7 × 10⁴ per g liver to rIFN-α–treated values of 2 × 10⁵ per g liver (Figure 5, b and d). Therefore, rIFN-α elicited a threefold amplification of NK cell yields in liver. Taken together, these results show that rIFN-α treatment promotes both MIP-1α production and accumulation of liver NK cells even in the absence of virus infection.

Requirement for MIP-1α in the rIFN-α induction of NK cell accumulation. To demonstrate the role of rIFN-α induction of MIP-1α in NK cell liver accumulation, the response was evaluated in MIP-1α⁺ and MIP-1α^– mice treated with vehicle or rIFN-α. Compared with vehicle-treated mice, MIP-1α⁺ mice demonstrated twofold increases in total cell yields (3 × 10⁶ to 7 × 10⁶ per g liver) and proportions of NK cells (11% to 19%) after rIFN-α treatment. In contrast, these parameters were not significantly affected after rIFN-α treatment of MIP-1α^– mice. Consequently, NK cell numbers were profoundly elevated in MIP-1α⁺ mice from 3 × 10⁵ per g liver after vehicle treatment to 1 × 10⁶ per g liver after rIFN-α administration, whereas the NK cell numbers for vehicle- and rIFN-α–treated MIP-1α^– mice remained comparable (Figure 5e). Hence, MIP-1α is required for the IFN-α/β–induced accumulation of NK cells in liver.

IFN-α/β induction of liver MIP-1α via cell delivery pathways during infection. Studies were conducted evaluating the requirement for cytokine function in trafficking of bone marrow–derived cells to liver and the contribution of these trafficking populations to liver MIP-1α production. Cells were fluorescently labeled with PKH26 and intravenously transferred into uninfected or MCMV-infected MIP-1α^– recipient mice. Bone marrow populations isolated from uninfected mice were used for cell transfers. Donor cells for analyses were prepared from untreated IFN-α/βR⁺ or IFN-α/βR^– mice, and transferred into MIP-1α^– recipient mice that were uninfected or infected with MCMV for 24 hours. Livers were harvested at 24 hours after cell transfer, i.e., 48 hours after MCMV infection. Samples were sectioned and analyzed by fluorescent microscopy, or homogenates were prepared, to measure MIP-1α production. Donor-derived cells were visible in sinusoidal cavities surrounding hepatocytes in both uninfected (Figure 6a) and infected (Figure 6, b and c) MIP-1α^– recipient mice. Although trafficking to these areas was observed in all sections, donor cell populations isolated from IFN-α/βR⁺ and transferred into infected mice (Figure 6, b and e) demonstrated a threefold induction in localized cells when compared with IFN-α/βR⁺ cells transferred into uninfected (Figure 6, a and d), or IFN-α/βR^– cells transferred into infected (Figure 6, c and f), MIP-1α^– recipients (Table 1). Thus, the trafficking of bone marrow–derived cells to the liver is induced in response to infection and dependent upon the ability of trafficking cells to respond to IFN-α/β effects.

Liver homogenates were prepared from all of the MIP-1α^– recipient mice to evaluate production of MIP-1α by trafficking cells. The chemokine was only detected in the samples from infected mice receiving bone marrow–derived cells from IFN-α/βR⁺ mice (Table 1). In contrast, MIP-1α protein levels in samples from MCMV-infected recipient mice with localized donor cells from IFN-α/βR^– mice were below the limit of detection (Table 1). Likewise, samples from all uninfected mice were also below the limit of detection. Because the MIP-1α had to come from the donor cells in these experiments, the results demonstrate that IFN-α/β can enhance local MIP-1α production by promoting recruitment of MIP-1α–producing cells.

IFN-α/β effects on macrophage trafficking and accumulation. As macrophages can localize in liver (1), experiments were performed to evaluate the roles of IFN-α/β functions for trafficking and accumulation of macrophages. Macrophages are F4/80–positive, and immigrating macrophage populations are defined by expression of both F4/80 and CD11b (21). Donor bone marrow–derived cells were prepared from untreated IFN-α/βR⁺ or IFN-α/βR^– mice, fluorescently labeled with PKH26, and transferred intravenously into IFN-α/βR⁺ or IFN-α/βR^– recipient mice treated with either vehicle or rIFN-α. Livers were harvested at 24 hours after cell transfer. Samples were sectioned and analyzed by fluorescent microscopy. Alternatively, liver leukocytes were prepared and analyzed for expression of F4/80 and CD11b to determine the numbers of donor-derived macrophages using flow cytometric and cell yield analyses. Donor-derived cells were evident in liver sinusoids from all samples (Figure 7, a–d). However, the transfer of cells from IFN-α/βR⁺ into rIFN-α–treated IFN-α/βR⁺ mice (Figure 7a) demonstrated a fivefold induction in number of total and a three- to fivefold induction in the number of macrophage donor-derived populations (Figure 7e) as compared with transfers into rIFN-α–treated IFN-α/βR^– or vehicle-treated IFN-α/βR⁺ mice (Figure 7, b and e). In contrast, donor cells isolated from IFN-α/βR^– and transferred into rIFN-α–treated IFN-α/βR⁺ (Figure 7c) or IFN-α/βR^– mice (Figure 7d) were limited in their ability to migrate into liver. The number of total and macrophage donor-derived cells did not change significantly when compared with those isolated from IFN-α/βR⁺ and transferred into vehicle-treated IFN-α/βR⁺ (Figure 7e) mice. Thus, the results demonstrate that IFN-α/β–mediated effects on both donor and recipient populations promote the migration of macrophages into livers.

To determine the effects of IFN-α/β on overall macrophage accumulation, the proportions and numbers of F4/80⁺CD11b⁺ cells were determined in liver leukocyte populations after either rIFN-α treatment or MCMV infection. Following administration of rIFN-α, the frequency of F4/80⁺ cells, and that of F4/80⁺ cells also expressing CD11b, were elevated twofold after rIFN-α as compared with those in mice receiving vehicle treatments (Figure 7f). The immigrating macrophage cell numbers increased from the vehicle-treated values of 1 × 10⁴ to 5 × 10⁴ per g liver (Figure 7g). Under the conditions of either moderate-dose (Figure 8a) or low-dose (Figure 8b) MCMV infections, the frequencies of F4/80⁺ cells increased approximately twofold and the frequencies of those also expressing CD11b increased to 83–87% from the uninfected values of 53–65% in IFN-α/βR⁺ mice. As the infection-induced total liver leukocyte numbers increased to 2 × 10⁶ per g liver after moderate dose and 3 × 10⁶ per g liver after low dose from the uninfected values of 1 × 10⁶ per g liver, IFN-α/βR⁺ mice had more-than-fivefold elevations in immigrating macrophage cell numbers at 48 hours after infection (Figure 8, a and b). In contrast, although the frequencies of F4/80⁺ cells did increase in infected IFN-α/βR^– mice, the frequencies of F4/80⁺ cells also expressing CD11b macrophage populations decreased two- to fourfold (Figure 8, a and b) compared with those in uninfected mice. Moreover, liver leukocyte yields from IFN-α/βR^– mice were only 9 × 10⁵ per g liver after moderate-dose and 1 × 10⁶ per g liver after low-dose infection, compared with the uninfected values of 1 to 2 × 10⁶ per g liver. Hence, the numbers of immigrating macrophages in IFN-α/βR^– mice were significantly lower after MCMV infection (Figure 8, a and b). Taken together, these results demonstrate that IFN-α/β mediates trafficking and accumulation of macrophages into liver.

Characterization of MIP-1α–producing cells. Macrophages are a known source of MIP-1α (1, 8). To determine whether trafficking macrophages contributed to production of MIP-1α, total liver leukocytes and enriched macrophages were prepared from C57BL/6 (Figure 8c) and 129 (Figure 8d) mice that were uninfected or infected with low-dose MCMV for 48 hours; they were then evaluated for their ability to release the chemokine. Following infection, total populations were induced to produce MIP-1α. However, the levels of MIP-1α produced by enriched trafficking macrophages, expressing both F4/80 and CD11b, were dramatically elevated over the levels produced by total leukocytes. The macrophages from infected C57BL/6 (Figure 8c) or infected 129 (Figure 8d) mice produced 12-fold or 22-fold more MIP-1α protein, respectively, when compared with total cells. Therefore, migrating macrophages are a major source of MIP-1α production in liver during MCMV infection.