Elsevier

Cellular Immunology

Volume 218, Issues 1–2, July–August 2002, Pages 74-86
Cellular Immunology

Plasmacytoid dendritic cells produce cytokines and mature in response to the TLR7 agonists, imiquimod and resiquimod

https://doi.org/10.1016/S0008-8749(02)00517-8Get rights and content

Abstract

The immune response modifiers, imiquimod and resiquimod, are TLR7 agonists that induce type I interferon in numerous species, including humans. Recently, it was shown that plasmacytoid dendritic cells (pDC) are the primary interferon-producing cells in the blood in response to viral infections. Here, we characterize the activation of human pDC with the TLR7 agonists imiquimod and resiquimod. Results indicate that imiquimod and resiquimod induce IFN-α and IFN-ω from purified pDC, and pDC are the principle IFN-producing cells in the blood. Resiquimod-stimulated pDC also produce a number of other cytokines including TNF-α and IP-10. Resiquimod enhances co-stimulatory marker expression, CCR7 expression, and pDC viability. Resiquimod was compared throughout the study to the pDC survival factors, IL-3 and IFN-α; resiquimod more effectively matures pDC than either IL-3 or IFN-α alone. These results demonstrate that imidazoquinoline molecules directly induce pDC maturation as determined by cytokine induction, CCR7 and co-stimulatory marker expression and prolonging viability.

Introduction

Dendritic cells (DC1) constitute heterogeneous populations of antigen presenting cells that are critical for bridging the innate and the adaptive immune responses [1], [2]. DC are found virtually in every tissue and organ. In the blood, DC can be sub-divided into two major populations, CD11c+ and CD11c[3], [4]. The CD11c+ population is thought to be myeloid-derived, therefore called myeloid DC (mDC). The CD11c population appears to be lymphoid-derived and constitutes a population of cells that can migrate from the blood directly to lymphoid tissues. The CD11c population are called plasmacytoid DC (pDC), because these cells have a morphology resembling plasma cells due to an extensive endoplasmic reticulum [5], [6]. Technically, pDC in the blood are precursor DC, unlike the mDC, because blood pDC require further differentiation to up-regulate CD80 and CD86, which is thought to occur in lymphoid tissue, before they develop the capacity to stimulate T-cell responses [7], [8], [9], [10].

The blood CD11c DC or pDC have recently been shown to be the major type I IFN-producing cells in response to virus infection [11], [12]. Type I IFNs are clinically important cytokines because of their use as adjuvants for anti-viral and anti-cancer therapy. In addition to their direct anti-viral and anti-proliferative affects, type I interferons have multiple affects on the immune system including up-regulation of MHC class I and II, enhanced co-stimulatory marker expression on DC, modulation of immunoglobulin production, synergizing with IL-12 to enhance IFN-γ production, and augmentation of NK and CTL responses [4], [13], [14], [15], [16], [17]. Thus, type I IFN contributes to both innate and adaptive immunity.

Potent inducers of IFN-α include the imidazoquinoline molecules. These molecules are classified as immune response modifiers (IRMs) and constitute a family of low molecular weight synthetic compounds that have broad anti-viral and anti-tumor activity in numerous animal models [18], [19], [20], [21], [22], [23], [24], [25]. One such molecule, imiquimod (Aldara, 5% cream), is currently available for the treatment of external genital and perianal warts caused by human papillomavirus [26]. Another molecule, resiquimod, has shown promise as a treatment for herpes simplex virus infection and is currently in phase III clinical studies for the treatment of genital herpes [27]. The immunological mechanism of action of resiquimod and imiquimod requires the activation of antigen presenting cells and production of various cytokines, particularly IFN-α, resulting in Th1-like immunity. In addition, resiquimod and imiquimod directly activate innate immune responses through a MyD88/TLR7-dependent pathway [28].

Earlier studies indicated that monocytes were the predominant IFN-α and pro-inflammatory cytokine-producing cells in the blood following imidazoquinoline stimulation [29]. Cell-depletion studies in vitro also implicated monocytes as the primary IFN-producing cells in the blood. Four lines of evidence suggested that we needed to re-investigate the question regarding which populations of cells produce IFN-α in response to imidazoquinoline molecules. First, DC recently have been shown to be composed of a multitude of subsets which produce different types of cytokines following activation. In response to viral infections, the pDC subset is the predominant IFN-α-producing cell in the blood [12]. Second, similar to viral infections, imidazoquinoline molecules induce the production of high levels of IFN-α from human peripheral blood. Third, the earlier published cell-depletion studies used antibody that was directed to the CD11c+ population of blood DC, thereby not eliminating the pDC subset [29]. Fourth, imiquimod- and resiquimod-induced cytokine production are MyD88/TLR7-dependent and pDC are the prominent TLR7-expressing DC subset [28], [30], [31]. Here, we evaluate the effects of imiquimod and resiquimod on pDC function, including cytokine production, co-stimulatory marker expression, CCR7 expression and viability.

Section snippets

Reagents

The TLR7 agonists resiquimod (R-848, S-28463), 4-amino-2-ethoxymethyl-α,α-dimethyl-1H-imidazo[4,5-c]quinoline-1-ethanol, MW=314.4, and imiquimod (R-837, S-26308), 1-(2-methylpropyl)-1H-imidazo[4,5-c]quinolin-4-amine, MW=243.3 were dissolved in dimethyl sulfoxide (DMSO, sterile cell culture grade, Sigma, St. Louis, MO) at a concentration of 12 mM and stored in aliquots at −20 °C. Resiquimod and imiquimod were added to cell cultures at various concentrations as described below. LPS (Salmonella

Resiquimod and imiquimod can activate NFκB through human TLR7

The mechanism of action of imidazoquinoline molecules involves, at least in part, TLR7. Resiquimod- and imiquimod-induced cytokine production were abrogated in TLR7-deficient mice; human TLR7 was shown as well to recapitulate resiquimod-induced NFκB activation in transiently transfected HEK293 cells [28]. Additionally, imiquimod can also activate NFκB-luciferase through human TLR7 in transiently transfected HEK293 cells, although not as robustly as resiquimod (Table 2). The minimum

Discussion

The results presented here demonstrate that the IRMs imiquimod and resiquimod can activate NFκB through TLR7 and can induce the production of type I IFN from pDC; these TLR7 agonists effectively induce pDC maturation. DC maturation is measured on a continuum rather than absolute criteria and the continuum of maturation is based on numerous criteria including cytokine production, co-stimulatory marker expression, CCR7 expression, and enhanced survival. Therefore, these data demonstrate that

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