A chemokine-to-cytokine-to-chemokine cascade critical in antiviral defense - PubMed (original) (raw)

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

T P Salazar-Mather et al. J Clin Invest. 2000 Apr.

Abstract

Macrophage inflammatory protein 1alpha (MIP-1alpha) promotes natural killer (NK) cell inflammation in livers during murine cytomegalovirus (MCMV) infections, and NK cell-produced interferon gamma (IFN-gamma) contributes to defense against MCMV infections. A specific role for local NK cell IFN-gamma production, however, has not been established. The importance of MIP-1alpha and NK cell-produced IFN-gamma in shaping endogenous immune responses and defense in different compartments was examined. MIP-1alpha deficiency profoundly decreased resistance to MCMV and was associated with dramatically reduced NK cell accumulation and IFN-gamma production in liver. MIP-1alpha-independent IFN-gamma responses were observed in serum and spleen, and infection-induced elevations in blood NK cell populations occurred in absence of the factor, but peak liver expression of another chemokine, the monokine induced by IFN-gamma (Mig), depended upon presence of MIP-1alpha, NK cells, and IFN-gamma. The Mig response was also important for viral resistance. Thus, serum cytokine responses are insufficient; MIP-1alpha is critical for NK cell migration and IFN-gamma delivery to mediate protection; and Mig induction in tissues is a downstream protective response resulting from the process. These results define a critical chemokine-to-cytokine-to-chemokine cascade required for defense during a viral infection establishing itself in tissues.

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Figures

Figure 1

Compartmental changes in NK cell populations. Blood (a and b) and liver (c and d) leukocytes were prepared from C57BL/6-MIP-1α+/+ (MIP-1α+) or C57BL/6-MIP-1α–/– (MIP-1α–) mice either uninfected or infected with MCMV for 48 hours. Leukocytes were analyzed by flow cytometry as described in Methods. Both the percentage (a and c) and absolute number (b and d) of NK1.1+CD3– cells per milliliter of blood or NK1.1+TCR-β– cells per entire liver are shown. Data are means ± SE (n = 3). Compartmental changes represent results from 1 of 3 repetitive experiments. Differences between control MIP-1α+ and MIP-1α– are significant, A_P_ < 0.01, B_P_ < 0.001.

Figure 2

IFN-γ production in serum, spleen, and liver. Serum samples (a) and spleen (b) or liver (c) homogenates were prepared from uninfected or MCMV-infected (at 24, 36, and 48 hours after infection) MIP-1α+ or MIP-1α– mice. IFN-γ protein was measured by ELISA. Each spleen homogenate data point consists of 6 animals tested individually. For liver homogenates, each uninfected and 24-hour data point consists of 6 animals, and each 36- and 48-hours data point consists of 9 animals, all tested individually. The means ± SE are shown. All samples from uninfected or 24-hour MCMV-infected mice were below the level of detection. Differences between MIP-1α+ and MIP-1α– are significant, A_P_ < 0.0005.

Figure 3

Effects of MIP-1α and IFN-γ on resistance to MCMV. (a and b) MIP-1α+, MIP-1α–, IFN-γ–/– (IFN-γ–), and MIP-1α– IFN-γ– mice were uninfected or infected with 5 × 104 PFU MCMV at times indicated. Spleen (a) and livers (b) were harvested and homogenized for viral titer determinations as described in Methods. Limit of detection for the assay was 2 log PFU per gram of tissue. Means ± SE (n = 3) are shown. (c) Mice were uninfected or infected with 105 PFU (high dose) MCMV and monitored twice daily for survival. Results represent 1 of at least 2 experiments. Data shown represent the mean ± SE (n = 6).

Figure 4

Requirements for IFN-γ and Mig gene expression. (a and b) Liver RNA was isolated from MIP-1α+, MIP-1α-, or IFN-γ– mice that were uninfected or infected with MCMV. (a) Total RNA was analyzed by Northern blot hybridization. Results represent 1 of 3 experiments. (b) Relative quantitative RT-PCR was carried out with Competimers for 18-S rRNA as internal controls. (c) Total liver RNA was prepared from MIP-1α+ and MIP-1α– mice administered control IgG (denoted as C) or anti-CD3 (α-CD3) at 3 hours before harvest. RNA was prepared and analyzed by relative quantitative RT-PCR as above. (d) Liver RNA was isolated from mice that were uninfected or infected with MCMV, with or without NK cell depletions. Mice were C57BL/6 or C57BL/6-SCID mice treated with control IgG (denoted as C), anti-NK1.1, or anti-AGM1 24 hours before infection. RNA was analyzed by relative quantitative RT-PCR as above. b–d show ethidium bromide–stained gel of amplified products, representing 1 of 3 experiments (see Table 1 for quantitation of results and Methods for details of procedures).

Figure 5

Induction of Mig protein expression. Livers were harvested from uninfected or 36 and 48-hour MCMV-infected MIP-1α+ and MIP-1α– mice, or 48-hour MCMV-infected C57BL/6-SCID mice. Tissue sections were prepared and immunohistochemically stained as described in Methods. The Mig protein is identified by dark blue precipitates. Tissues are counterstained with methyl green. Results shown are from (a) MIP-1α+ mice after 36 hours of MCMV infection, (b) MIP-1α– mice after 36 hours of MCMV infection, (c and e) MIP-1α+ mice after 48 hours of MCMV infection, (d and f) MIP-1α– mice after 48 hours of MCMV infection, (g) uninfected MIP-1α+, and (h) C57BL/6-SCID mice after 48 hours of MCMV infection. Photographs were taken at magnifications of ×31.25 (a–d, g, h) or ×125 (e, f).

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A chemokine-to-cytokine-to-chemokine cascade critical in antiviral defense - PubMed (original) (raw)