Innate immune response of the human host to exposure with herpes simplex virus type 1: in vitro control of the virus infection by enhanced natural killer activity via interleukin-15 induction

Abstract

Infections with herpes simplex virus type 1 (HSV-1) in humans and in animal models are accompanied by enhanced natural killer (NK) activity. In vitro, HSV-1 also enhances the NK activity of human peripheral blood mononuclear cells (PBMC). The molecular basis of this enhanced NK activity, however, is not well characterized. We investigated the role of human interleukin-15 (IL-15) in this phenomenon and report here that HSV-1-mediated enhanced NK activity was abrogated by neutralizing antibodies for IL-15 but not for other cytokines (i.e., IL-2, IL-12, gamma interferon [IFN-gamma], tumor necrosis factor alpha, or IFN-alpha). Anti-CD122 antibodies which block signaling through IL-2 receptor beta chain, and therefore neutralize the effects of IL-15 (and IL-2), also abrogated this enhancement. Furthermore, HSV-1 increased the levels of IL-15 mRNA and the production of IL-15 in HSV-1-infected PBMC cultures. The neutralization of IL-15 in cocultures of PBMC with HSV-1-infected cells significantly increased HSV-1 production. These results strongly suggest a role for IL-15 in the HSV-1-mediated in vitro enhancement of NK activity and in the PBMC-mediated suppression of HSV-1 replication.

FIG. 1

HSV-1 exposure induces enhancement of NK activity of PBMC. (A) Four million PBMC were incubated with HSV-1 or with mock virus preparation at 37°C for 1 h, washed, and incubated at 37°C in 5% CO₂. After 24 h, their NK activity was determined at a target/effector cell ratio of 1:20 against 10^{4 51}Cr-labeled K562 cells. Data represent the average NK activity (percent lysis) ± SE of PBMC from three different donors (A, B, and C) with and without (mock) treatment with HSV-1. (B) NK assays were set up in triplicate in 96-well microculture plates using PBMC without treatment with HSV-1. Different doses of HSV-1 (0 to 100 μl) were added to these assays, and NK activity was determined 16 h later. The data show that the optimum dose of virus to induce maximum lysis was 50 μl. Note that 100 μl of the virus itself (without PBMC) did not cause significant lysis of the target cells (bar with asterisk). (C) Average lysis ± SE from three different donors (A, B, and C) after the addition of 50 μl of HSV-1 or mock virus preparation to NK assays (as described for panel B). (D) PBMC were incubated with purified HSV-1 or with similar noninfectious UV- or heat-treated viral preparations, and NK activities were determined 24 h later as described for panel A. Both UV- and heat-inactivated viruses also enhanced the NK activities of PBMC as did the concentrated infectious HSV-1.

FIG. 2

PBMC were incubated with HSV-1, washed, and incubated at 37°C in 5% CO₂ as described for Fig. 1A. NK activity against ⁵¹Cr-labeled K562 targets was determined at different time points postincubation. Note the peak increase in NK activity in HSV-treated PBMC at 24 h after the treatment.

FIG. 3

The culture SN from HSV-1-treated PBMC increases NK activity of untreated PBMC. (A) PBMC (4 × 10⁶) were treated with HSV-1, washed, and incubated at 37°C for 24 h as described in the legend to Fig. 1A. Their SN were collected and filtered through 0.1-μm-pore-size filters. Freshly isolated PBMC were incubated at 37°C in this SN or with SN from the mock-treated PBMC for 24 h and then used as effectors in NK assays. The effects of these SN from three different donors on the NK activity of the untreated PBMC are shown. A, NK activity of PBMC incubated for 24 h in culture medium; B to D, NK activity of the same PBMC after incubation with SN from mock-treated PBMC of three donors; E to G, NK activity of the PBMC after incubation with SN from HSV-treated PBMC of the same three donors, respectively. (B) The addition of 50 μl of 10-fold-concentrated culture SN from HSV-treated, but not from mock-treated, PBMC to NK assays enhances the NK activity of freshly isolated untreated PBMC. A, NK activity of PBMC without SN; B to D, NK activity of PBMC with the addition of SN from the mock-treated PBMC of three different donors; E to G, NK activity of PBMC with the addition of SN from HSV-treated PBMC of the same three donors, respectively.

FIG. 4

IL-15-neutralizing antibodies inhibit the HSV-1-mediated increase in the NK activity of PBMC. PBMC were treated with HSV-1 and incubated in the presence of different cytokine neutralizing antibodies. Their NK activities were determined 24 h later. The effects of neutralization of different cytokines on the HSV-1-mediated increase in NK activity of PBMC are shown. A, mock-treated PBMC; B, HSV-treated PBMC; C to H, HSV-infected PBMC incubated in the presence of neutralizing antibodies for cytokines IL-12, TNF-α, IFN-γ, IL-15, control, and IFN-α, respectively. Note that only significant (P < 0.05) inhibition of the HSV-mediated enhancement of NK activity was caused by IL-15-neutralizing antibodies (compare F with B).

FIG. 5

Effects of anti-CD122 antibodies on HSV-induced enhancement of NK activity. PBMC were mock treated or treated with HSV-1, washed and incubated in the presence of anti-CD122 or control antibodies for 24 h, and used as effectors in NK assays. The data show the average ± SE NK activities of these PBMC. A, mock-treated PBMC; B, HSV-treated PBMC; C, HSV-treated PBMC incubated in the presence of anti-CD122 antibodies; D, HSV-treated PBMC incubated in the presence of control antibodies; E, HSV-treated PBMC incubated in the presence of anti-IL-15 antibodies.

FIG. 6

HSV-1 increases IL-15 mRNA levels in PBMC. (A) Four million PBMC were incubated with HSV-1. Total RNA was extracted from the PBMC at the indicated time points, reverse transcribed, and used in PCR for amplification of the IL-15 or β-actin cDNA segments as described in Materials and Methods. The PCR products were validated on Southern blots using gene-specific ³²P-labeled probes. The amplified transcripts from the PBMC of two different donors (1 and 2) are shown. The bands in rows A and B show RT-PCR products of IL-15 and β-actin, respectively. Lanes a to d represent 0, 4, 8, and 12 h of incubation with the virus. (B) Densitometric analysis of the IL-15 transcripts expressed as a ratio with β-actin transcripts at different time points after incubation of the PBMC with HSV-1.