Measles virus infection of alveolar macrophages and dendritic cells precedes spread to lymphatic organs in transgenic mice expressing human signaling lymphocytic activation molecule (SLAM, CD150)

Abstract

Recent studies of primate models suggest that wild-type measles virus (MV) infects immune cells located in the airways before spreading systemically, but the identity of these cells is unknown. To identify cells supporting primary MV infection, we took advantage of mice expressing the MV receptor human signaling lymphocyte activation molecule (SLAM, CD150) with human-like tissue specificity. We infected these mice intranasally (IN) with a wild-type MV expressing green fluorescent protein. One, two, or three days after inoculation, nasal-associated lymphoid tissue (NALT), the lungs, several lymph nodes (LNs), the spleen, and the thymus were collected and analyzed by microscopy and flow cytometry, and virus isolation was attempted. One day after inoculation, MV replication was documented only in the airways, in about 2.5% of alveolar macrophages (AM) and 0.5% of dendritic cells (DC). These cells expressed human SLAM, and it was observed that MV infection temporarily enhanced SLAM expression. Later, MV infected other immune cell types, including B and T lymphocytes. Virus was isolated from lymphatic tissue as early as 2 days post-IN inoculation; the mediastinal lymph node was an early site of replication and supported high levels of infection. Three days after intraperitoneal inoculation, 1 to 8% of the mediastinal LN cells were infected. Thus, MV infection of alveolar macrophages and subepithelial dendritic cells in the airways precedes infection of lymphocytes in lymphatic organs of mice expressing human SLAM with human-like tissue specificity.

FIG. 1.

Analysis of hSLAM expression in leukocytes from the mediastinal LNs of Ifnar^ko-SLAMGe mice. Cells were stained with antibodies specific for B cells (B220⁺) (A), T cells (CD3⁺) (B), or macrophages (CD11c^low Mac-1^high) (C). Ifnar^ko-CD46Ge mice were used as negative controls (right, blue lines). % of Max is a measure of the number of cells normalized to the same total number in each sample.

FIG. 2.

Analysis of SLAM expression in leukocytes from the lungs of Ifnar^ko-SLAMGe mice. (A and B) Lymphocytes were gated and sorted with antibodies against B cells (B220⁺) or T cells (CD3⁺), respectively. For AM (C), total live cells were stained with three antibodies and sorted for the combination CD11c^high MHCII^low Mac-1^low. Ifnar^ko-CD46Ge mice were used as negative controls. For histograms, % of Max is a measure of the number of cells normalized to the same total number in each sample. FSC, forward scatter.

FIG. 3.

Characterization of wtMV-infected cells in the lungs 1 day after mouse inoculation. (A and B) Frozen sections of mouse lung stained with an anti-N antibody and counterstained with hematoxylin. N protein was visualized with red chromogen. Arrows indicate positive cells, which localize in the airway lumen. (C to H) Identification of the cell types accumulating MV N by confocal micrograph analysis of lung frozen sections. N protein was visualized with Alexa Fluor 488 (green). The specific cell markers indicated on the top right of each panel were visualized with Alexa Fluor 594 (red). Nuclei were counterstained in blue with DAPI. Scale bars represent 10 μm.

FIG. 4.

Analysis of hSLAM expression in AM before and after wtMV infection. AM from noninfected Ifnar^ko-CD46Ge mice (A), mock-infected Ifnar^ko-SLAMGe (B), or MV-infected Ifnar^ko-SLAMGe mice (C). Cells were gated for CD11c^high (left), MHCII^low (center left), and Mac-1^low (center right) and analyzed for infection (GFP) versus SLAM expression (right). Effect of MV infection on hSLAM expression in AM (D). FSC, forward scatter. Ifnar^ko-CD46Ge mice were used as negative controls to set the quadrants.

FIG. 5.

wtMV infection levels in lymphatic organs. Data were obtained 3 days after IN inoculation (A), 3 days after IP inoculation (B), or 6 days after IP inoculation (C). Each dot represents a single mouse. Group average is represented by a horizontal bar, and detection limit by a dotted line. Man, mandibular LN; Med, mediastinal LN; Mes, mesenteric LN; Ing, inguinal LN; Spl, spleen.