Elsevier

Human Immunology

Volume 65, Issue 2, February 2004, Pages 104-113
Human Immunology

Identification and characterization of intestinal peyer's patch interferon-α producing (plasmacytoid) dendritic cells

https://doi.org/10.1016/j.humimm.2003.10.006Get rights and content

Abstract

Recently, a subset of murine dendritic cells (DC) has been identified that resembles human plasmacytoid (pDC) the principal interferon-α (IFN-α) producing cells in blood. In this study, C57BL/10 (B10;H2b) mice were treated with fms-like tyrosine 3 kinase Ligand (Flt3L; 10 μg/d; i.p.; 10 days) that expands DC selectively in vivo. Putative pDC (CD11c+B220+) were identified in the subepithelial dome and in interfollicular regions of intestinal Peyer's patches (PP) from both normal and Flt3L-treated animals. Freshly-isolated, immunobead-purified CD11c+ DC from PP were flow-sorted to obtain lineage (CD11bCD19) CD11c+ B220+ DC (purity > 96%). Flow cytometric analysis revealed that these sorted PPpDC were negative for surface markers associated with myeloid DC (CD11b) and expressed only low levels of the “lymphoid-related” DC marker CD8αα+. They expressed low levels of costimulatory molecules and moderate MHC class II. They proved weak stimulators of naı̈ve allogeneic (C3H; H2k) T-cell proliferation. Cytospin preparations of sorted CD11c+B220+ cells revealed plasmacytoid morphology similar to that of human pDC. Immunocytochemistry and enzyme immunoassay revealed that, within 24-hour culture with Herpes simplex virus (10 p.f.u./cell), a subpopulation of stimulated (but not unstimulated) CD11c+B220+ DC produced and secreted IFN-α. This novel DC subset may play important roles in innate and adaptive immune responses of the gut and in the regulation of mucosal immune reactions.

Introduction

Most antigens (Ag) gain entry to the body via mucosal surfaces. They are taken up and processed by professional Ag-presenting cells (APC), in particular dendritic cells (DC) that transport the Ag via lymphatics to T-cell areas of regional lymphoid organs. Cells within gut-associated lymphoid tissue (GALT) are able to discriminate effectively between innocuous and harmful Ags, and either immunologic unresponsiveness (oral tolerance) or local and systemic immune reactivity is induced 1, 2, 3. Peyer's patches (PP) are organized lymphoid aggregates within the wall of the small intestine. They are considered the main sites of the GALT where microbial and food Ags are presented to immune effector cells. DC are uniquely well-equipped professional APCs that are believed to play key roles both in the initiation and modulation of mucosal immune responses 2, 4, 5.

In the murine PP, three distinct subsets of DC have been identified based on their differential expression of specific cell surface markers and their characteristic localization 6, 7, 8. All subsets express CD11c and major histocompatibility complex (MHC) class II Ags, but differ in their expression of CD8α and CD11b. CD11c+CD8α+ “lymphoid-related” DC are localized in the interfollicular regions of PP. Myeloid DC (CD11c+CD11b+) are found under the follicle-associated epithelium in the subepithelial dome of PP, where they are ideally situated for the uptake of luminal Ags transported by M cells [9]. A third population of “double-negative” (CD8αCD11b) PPDC has also been described, in both sites [7]. Recent data support the view that lymphoid-related or double-negative PPDC induce T helper 1 (Th1) cell differentiation, whereas myeloid PPDCs are better able to skew T-cell responses to Th2 differentiation [7].

Recently, a novel subset of murine CD11c+ cells that expresses high levels of surface B220 has been identified in spleen, lymph nodes, and bone marrow 10, 11. Unlike other DC, these CD11c+B220+ cells exhibit a plasmacytoid morphology and, when challenged with virus, produce type-1 interferon-α (IFN-α). Based on these morphologic and functional features, this DC subset has been proposed as the murine equivalent of human plasmacytoid DC (pDC) [12]. This DC population is able to induce a non-anergic state of T-cell unresponsiveness that involves the differentiation of T-regulatory (T reg) cells with ability to inhibit Ag-specific T-cell proliferation. pDC may represent a subset of potentially tolerogenic DC 13, 14. In this study, we describe a novel CD11c+B220+ DC subset within the PP of mice treated with fms-like tyrosine-3 kinase ligand (Flt3L), a potent endogenous hematopoietic growth factor that expands DC in vivo without modifying their activation status 8, 15. These cells appear as immature plasmacytoid cells when freshly isolated, but develop a distinct dendritic morphology when incubated overnight with IL-3 and anti-CD40 monoclonal antibody (mAb). They also exhibit very low T-cell stimulatory activity when freshly isolated and produce IFN-α when infected with herpes simplex virus (HSV) [16]. These characteristics of CD11c+B220+ PPDC are similar to those reported for human pDC. PPpDC may play an important role in the regulation of mucosal immune reactivity, including antiviral and alloimmune responses.

Section snippets

Experimental animals

Ten- to 12- week-old C57BL/10 (B10; H2Kb, IAb, IE) and C3H/HeJ (C3H; H2Kk, IAk, IEk) mice were purchased from The Jackson Laboratory (Bar Harbor, ME, USA) and maintained in the specific pathogen-free Central Animal Facility of the University of Pittsburgh Medical Center. They received Purina rodent chow (Ralstan Purina, St. Louis, MO, USA) and tap water ad libitum. Experiments were conducted in accordance with the National Institutes of Health Guide for use and care of laboratory animals and

Flt3L administration expands PP CD11c+ DC without modifying DC subset localization

Treatment of mice with the endogenous hematopoietic growth factor Flt3L expands DC in lymphoid and nonlymphoid tissues, without modifying their functional state and overcomes the problem of their scarcity in normal tissues [8]. We first examined the influence of Flt3L on numbers of CD11c+ in PP using immunofluorescence microscopy and flow cytometric analysis. Sections were stained for the DC marker CD11c, using anti-CD11c mAb (red) alone or in combination with mAbs against CD11b, CD8α, or B220

Discussion

In this study we have used immunophenotypic, morphologic, and functional analyses to identify and characterise CD11c+B220+ DC isolated from C57BL/10 mouse PP. The hematopoietic growth factor Flt3L that binds to the Flk2/Flt3 receptor (CD135) is a valuable tool for the study of DC in situ. It does not induce their activation 8, 15, and was used in this investigation to expand DC in vivo. This growth factor has already been reported by others to regulate pDC generation in vivo because

Acknowledgements

We thank the Immunex Corporation (now Amgen) for providing Flt3L; Dr. P. Toby Coates, Mr. J. William Shufesky, Mr. F Jason Duncan, and Ms. Audrey Lau for helpful discussion and suggestions; Ms. Alison Logar for assistance with flow sorting and analysis; Dr. Antonio Francavilla (University of Bari, Italy) for support and encouragement; and Ms. Miriam Meade for skillful manuscript preparation. The study was supported by National Institutes of Health grants R01 DK49745, R01 AI41011, U01 AI51698

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