Macrophage inflammatory protein 3alpha deficiency in atopic dermatitis skin and role in innate immune response to vaccinia virus

Abstract

Background: Patients with atopic dermatitis (AD) are prone to disseminated viral skin infections and therefore are not vaccinated against smallpox because of potential complications. Macrophage inflammatory protein 3alpha (MIP-3alpha) is a C-C chemokine expressed by keratinocytes that exhibits antimicrobial activity against bacteria and fungi; however, its role in antiviral innate immunity is unknown.

Objective: Evaluate the level of MIP-3alpha in AD skin and its role in the innate immune response to vaccinia virus (VV).

Methods: Macrophage inflammatory protein 3alpha levels were evaluated using real-time RT-PCR, immunodot-blot, and immunohistochemistry. The antiviral activity of MIP-3alpha was determined using a standard viral plaque assay.

Results: Macrophage inflammatory protein 3alpha gene expression was significantly (P < .01) decreased in AD skin (0.21 +/- 0.05 ng MIP-3alpha/ng glyceraldehyde-3-phosphate dehydrogenase) compared with psoriasis skin (0.67 +/- 0.13). This was confirmed at the protein level using immunohistochemistry. We further demonstrate that T(H)2 cytokines downregulate MIP-3alpha expression. The importance of MIP-3alpha in the innate immune response against VV was established by first demonstrating that MIP-3alpha exhibits activity against VV. Second, VV replication was significantly increased (P < .01) in keratinocytes treated with an antibody to neutralize MIP-3alpha.

Conclusion: The current study demonstrates that MIP-3alpha exhibits antiviral activity against VV and demonstrates the importance of MIP-3alpha in the innate immune response against VV. In addition, AD skin is deficient in MIP-3alpha, in part because of the overexpression of T(H)2 cytokines in AD skin.

Clinical implications: MIP-3alpha deficiency in AD skin contributes to patients' increased propensity toward eczema vaccinatum. Increasing MIP-3alpha or neutralizing T(H)2 cytokines could prevent adverse reactions in patients with AD after smallpox vaccination.

Figure 1

MIP-3α gene expression is decreased in AD, as compared to psoriasis, skin. RNA was isolated from the skin of nine normal subjects, nine AD, and six psoriasis patients and the levels of MIP-3α gene expression were evaluated by real-time RT-PCR as described in the methods section. Data are expressed as the mean ± SEM.

Figure 2

MIP-3α protein expression is decreased in AD, as compared to psoriasis, skin. A. Paraffin embedded skin biopsies were cut into 5 μm sections and stained for MIP-3α. Representative images are shown at 200X magnification evaluated from six AD, six psoriasis and six normal subjects. B. The intensity of the immunostaining was visually scored on a scale from 0 to 8, where 0 indicated no staining, 1 = 0 – 12.5% staining, 2 = 12.5 – 25%, with a stepwise increase by 12.5% to 8 = 100. *indicates statistical significance of p<0.05.

Figure 2

MIP-3α protein expression is decreased in AD, as compared to psoriasis, skin. A. Paraffin embedded skin biopsies were cut into 5 μm sections and stained for MIP-3α. Representative images are shown at 200X magnification evaluated from six AD, six psoriasis and six normal subjects. B. The intensity of the immunostaining was visually scored on a scale from 0 to 8, where 0 indicated no staining, 1 = 0 – 12.5% staining, 2 = 12.5 – 25%, with a stepwise increase by 12.5% to 8 = 100. *indicates statistical significance of p<0.05.

Figure 3

MIP-3α is induced by VV and down-regulated by Th2 cytokines. Primary keratinocytes were stimulated with 0.1 pfu/cell of VV in the presence and absence of IL-4 (50 ng/ml) and IL-13 (50 ng/ml) for 24 hours. MIP-3α gene expression was evaluated by real-time RT-PCR. Data are expressed as the mean ± SEM with an n=6.

Figure 4

Quantification of MIP-3α protein levels in skin biopsies from normal subjects, lesional AD skin, and lesional psoriasis skin. Protein was isolated from the skin of six normal subjects, six AD, and six psoriasis patients and the levels of MIP-3α protein were evaluated by immuno-dot blot. Data are expressed as the mean ± SEM

Figure 5

MIP-3α exhibits anti-viral activity against VV. VV was incubated with varying concentrations of MIP-3α for 24 hours and then evaluated using a standard viral plaque assay. Data are expressed as the mean ± SEM with an n=6.

Figure 6

Neutralization of MIP-3α augments VV replication. HaCaT cells were stimulated with 0.1 pfu/cell of VV for 24 hours in the presence and absence of 1 μg/ml of anti-MIP-3α. VV gene expression was evaluated by real-time RT-PCR. Data are expressed as the mean ± SEM with an n=6.