Varicella-zoster virus transfer to skin by T Cells and modulation of viral replication by epidermal cell interferon-alpha

Abstract

Primary infection with varicella-zoster virus (VZV) causes the characteristic syndrome of varicella, or chickenpox. Experiments in severe combined immunodeficiency mice with human skin grafts (SCIDhu mice) indicate that VZV infection of T cells can mediate transfer of infectious virus to skin. VZV-infected T cells reached epithelial sites of replication within 24 h after entering the circulation. Memory CD4+ T cells were the predominant population recovered from skin in SCIDhu mice given uninfected or infected mononuclear cells, suggesting that immune surveillance by memory T cells may facilitate VZV transfer. The increased susceptibility of memory T cells to VZV infection may further enhance their role in VZV pathogenesis. During VZV skin infection, viral gene products down-regulated interferon-alpha to permit focal replication, whereas adjacent epidermal cells mounted a potent interferon-alpha response against cell-cell spread. Interleukin-1alpha, although activated in VZV-infected cells, did not trigger expression of endothelial adhesion molecules, thereby avoiding early recruitment of inflammatory cells. The prolonged varicella incubation period appears to represent the time required for VZV to overcome antiviral responses of epidermal cells and generate vesicles at the skin surface. Modulation of VZV replication by cutaneous innate immunity may avoid an incapacitating infection of the host that would limit opportunities for VZV transmission.

Figure 1.

Transfer of VZV by CD3⁺ T cells. Column-purified CD3⁺ T cells from tonsils were cocultured with VZV-infected HEL cells for 48 h before i.v. transfusion into SCID mice with human skin xenografts. Skin implants were harvested at various times after T cell transfer, snapped frozen, and sectioned. (A) Human fetal skin xenografts differentiated in SCID mice at 4 wk expressed human CD31 on the endothelial cells (magnification: 100×; insert, 200×). (B) 15–30% of tonsillar T cells used to inject SCIDhu mice expressed VZV glycoproteins as shown in a representative flow cytometric analysis. (C) CD3⁺ T cells were detected around hair follicles and along basement membranes 24 h after i.v. injection (left, arrows; insert, rabbit IgG control); VZV-induced cytopathology was shown by hematoxylin and eosin (H & E) stain 21 d after T cell transfer compared with uninfected control (middle) and by anti-VZV IgG (brown, top right; bottom right, VZV nonimmune IgG control) (magnification: left, 400×; middle and right, 100×).

Figure 2.

IL-1α expression in VZV-infected and uninfected epidermal cells. In this representative figure, formalin-fixed, paraffin-embedded sections of VZV-infected skin xenografts were stained with anti-VZV IgG, detected with DAB chromagen (brown) as shown in the left panel (magnification, 100×). Serial sections of these samples were stained with anti–IL-1α and detected with DAB as illustrated in the middle and right panels (magnification, 630×). IL-1α expression was analyzed in cells expressing VZV proteins as shown within the area outlined in the left panel and in neighboring uninfected epidermal cells in the same section. IL-1α was activated and translocated to the nuclei of VZV infected cells (middle) but showed a diffuse cytoplasmic stain in neighboring uninfected cells (right).

Figure 3.

IFN-α expression and Stat1 phosphorylation in VZV-infected and uninfected epidermal cells. Formalin- fixed, paraffin-embedded sections of VZV-infected skin xenografts were pretreated and double stained with anti-VZV IgG, detected with DAB (brown), and anti–IFN-α or anti-pStat1 antibody detected with Vector VIP (purple). Sections are shown at magnification 200×. (A) Infected cells were identified by VZV protein (brown) expression. In double labeled skin sections, IFN-α (purple) expression was prominent in adjacent uninfected cells but not in VZV-infected cells (top) compared with sections stained with rabbit IgG as a control for IFN-α (bottom). (B) In double labeled sections, phosphorylated Stat1 (purple) was up-regulated in adjacent uninfected cells but absent in VZV-infected cells (top); pStat1 was not detected in uninfected skin (bottom right) compared with rabbit IgG control stain (bottom left).

Figure 4.

Blocking of IFN-α signaling enhanced VZV replication in skin. Skin xenografts were infected with VZV by direct inoculation in mice treated with anti-IFNα/βRβ antibody or controls and harvested at day 7 after infection. Half of the skin implant was fixed in 4% paraformaldehyde for histological analysis, and the other half was disassociated into cell suspension to measure virus titers by infectious focus assay. (A and B) Shown are representative results from skin sections examined at 7 d (large arrow, VZV lesion); phosphorylated Stat1 (purple; small arrow) was translocated to nuclei in adjacent cells but not within VZV lesions (magnification, 200×). (C and D) Skin lesions shown by staining with anti-VZV IgG (purple) were larger at day 7 in animals given anti-IFNα/βRβ antibody (right) than controls (left) (magnification, 100×). (E) Mean VZV titers in four skin xenografts from two mice were 10-fold higher in anti-IFNα/βRβ antibody-treated animals compared with controls (P < 0.05, unpaired t test).

Figure 5.

Expression of adhesion molecules by cutaneous endothelial cells in VZV-infected skin xenografts and biopsies of VZV skin lesions. Serial frozen sections of VZV-infected skin xenografts (left) or lesion biopsies from patients with varicella or herpes zoster (right) were stained with antibodies against adhesion molecules expressed by endothelial cells. Signals were detected with Vector VIP (purple) and counterstained with methyl green. Representative results from one of three VZV-infected skin xenografts and one of five VZV lesion biopsies are shown for expression of CD31, E-selectin, ICAM-1, and VCAM-1. E-selectin, ICAM-1, and VCAM-1 were highly expressed by CD31-positive dermal endothelial cells in patient lesion biopsies but were not detected in VZV infected skin xenografts (asterisk, VZV skin lesion). Magnification, 100×.

Figure 6.

A model of the pathogenesis of primary VZV infection. T cells within the local lymphoid tissue of the respiratory tract may become infected by transfer of VZV from its initial site of inoculation in respiratory epithelial cells. T cells may then transport the virus to the skin immediately and release infectious VZV. The remainder of the 10–21-d incubation period appears to be the interval required for VZV to overcome the innate IFN-α response in enough epidermal cells to create the typical vesicular lesions containing cell-free virus at the skin surface. The signaling of enhanced IFN-α production in adjacent skin cells may prevent a rapid, uncontrolled cell–cell spread of VZV. Secondary “crops” of varicella lesions may result when T cells traffic through early stage cutaneous lesions become infected and produce a secondary viremia. Intact host immune responses appear to be required to trigger up-regulation of adhesion molecules, facilitating the clearance of VZV by adaptive immunity.