Cytomegalovirus MCK-2 controls mobilization and recruitment of myeloid progenitor cells to facilitate dissemination

Abstract

Murine cytomegalovirus encodes a secreted, pro-inflammatory chemokine-like protein, MCK-2, that recruits leukocytes and facilitates viral dissemination. We have shown that MCK-2-enhanced recruitment of myelomonocytic leukocytes with an immature phenotype occurs early during infection and is associated with efficient viral dissemination. Expression of MCK-2 drives the mobilization of a population of leukocytes from bone marrow that express myeloid marker Mac-1 (CD11b), intermediate levels of Gr-1 (Ly6 G/C), platelet-endothelial-cell adhesion molecule-1 (PECAM-1, CD31), together with heterogeneous levels of stem-cell antigen-1 (Sca-1, Ly-6 A /E). Recombinant MCK-2 mediates recruitment of this population even in the absence of viral infection. Recruitment of this cell population and viral dissemination via the bloodstream to salivary glands proceeds normally in mice that lack CCR2 and MCP-1 (CCL2), suggesting that recruitment of macrophages is not a requisite component of pathogenesis. Thus, a systemic impact of MCK-2 enhances the normal host response and causes a marked increase in myelomonocytic recruitment with an immature phenotype to initial sites of infection. Mobilization influences levels of virus dissemination via the bloodstream to salivary glands and is dependent on a myelomonocytic cell type other than mature macrophages.

Figure 1.

Schematic representation of mutant virus genomes. The top line represents the K181⁺ strain MCMV genome with nucleotide numbers (GenBank accession number U68299). The MCMV ie1/ie2/ie3 transcriptional enhancer (hatched box) and transcripts (solid arrows) encoded by the wild-type viruses also are shown in the expanded region. Open boxes depict the positions of ORFs m131 and m129 that encode MCK-2. The mutations introduced into recombinant viruses RM461, RM427⁺, RM4503, and RM4511 are depicted below the transcripts. The lacZ insertion in RM461 or RM427⁺ (open box) and the EGFP-puro insertion in RM4503 or RM4511 (open box) are depicted below the map. Two nucleotide point mutations were introduced into RM4511 to alter the conserved CC chemokine motif in MCK-2 (C₂₇R and C₂₈G; 14).

Figure 2.

Markers and cytology of myeloid cells at sites of infection. (A) FCM analysis on medium (left), mck-mutant RM461-infected (middle), and mck-expressing RQ461 (right). Percentages of cells with each expression pattern are indicated in the Mac-1^-/Gr-1⁺, Mac-1⁺/Gr-1⁺, and Mac-1⁺/Gr-1^- quadrants. The Mac-1⁺/Gr-1^int population is indicated by a dashed box. FP leukocytes were prepared from groups of 3 BALB/c mice at 48 hours after inoculation (inoculum of 1 × 10⁶ PFUs). (B) May-Grunwald-Giemsa-stained FCM purified cells from pooled FPs (20 mice) at 48 hours after inoculation (inoculum of 1 × 10⁶ PFUs), comparing 4 examples from the Mac-1⁺/Gr-1^int population (left) and 4 examples from the Mac-1^-/Gr-1^- population (right) (× 1000). Mac-1⁺/Gr-1^int cells were prepared by MACS separation, followed by FACSvantage sorting, and Mac-1^-/Gr-1^- cells were from the initial MACS separation step. (C) Summary data for the frequency of Mac-1⁺/Gr-1^int cells of FP leukocytes. The mean (bars) with standard deviation (error bars) of 3 independent experiments is indicated.

Figure 3.

FCM analysis of N-Met-MCK-2 recruited Mac-1⁺/Gr-1^int myeloid cells. (A) N-Met-MCK-2-induced swelling. BALB/c mice were injected into FPs 4 times with 200-ng doses of N-Met-MCK-2 (♦) or PBS carrier (⋄) using groups of mice as indicated by arrowheads below the graph. Standard deviation of mean swelling is indicated by error bars. (B) FCM analysis of cells recovered from FPs prepared from 2 independent experiments using groups of 4 (experiment 1) or 5 (experiment 2) BALB/c mice at 3 hours after the fourth N-Met-MCK-2 injection. FCM analysis and layout is described in the legend to Figure 2.

Figure 4.

Acquisition of virus by Mac-1⁺/Gr-1⁺ cells. (A) ICA on total FP leukocytes collected from 10 mice at 48 hours after inoculation with RQ461 or RM461 (inoculum of 1 × 10⁶ PFUs) shown as geometric mean percentage virus-positive FP cells ± standard deviation of the geometric mean (error bars). (B) FCM analysis of mck-expressing RM4503-infected infiltrates prepared from independent groups of 5 BALB/c mice at 72 hours after inoculation (inoculum of 1 × 10⁶ PFUs). Bottom left panel shows the Mac-1 and Gr-1 staining pattern for this population, and the 3 adjacent panels show FCM analysis of GFP in quadrants R2, R3, and R4, as indicated by arrows. A solid line indicates the histogram for RM4503, and a dashed line indicates the histogram for control RQ461-infected cells. (C) FCM analysis of mck-mutant RM4511-infected FP infiltrates prepared from 5 BALB/c mice at 72 hours after inoculation (inoculum of 1 × 10⁶ PFUs). Bottom left panel shows the Mac-1 and Gr-1 staining pattern for this population, and the 3 adjacent panels show FCM analysis for GFP in quadrants R2, R3, and R4. A solid line indicates the histogram for RM4511, and a dashed line indicates the histogram for control RM461-infected cells. The percentage of GFP⁺ cells is indicated above the brackets in each of the panels.

Figure 5.

Time course of Mac-1⁺/Gr-1^int cell appearance following MCMV infection. Leukocytes were prepared from FPs (A) and PB (B) of groups of 5 infected BALB/c mice on days 0, 1, 2, 3, and 5 after inoculation with mck-expressing RQ461 or mck-mutant RM461 (inoculum of 1 × 10⁶ PFUs). Mac-1⁺/Gr-1^int cell populations are indicated by the boxed areas. (B) An arrow in the day 3 sample identified the peak in mobilization of the Mac-1⁺/Gr-1^int cells into the bloodstream. (C) Summary of the kinetics of mobilization of Mac-1⁺/Gr-1^int cells into PB and recruitment into FPs.

Figure 6.

FCM light-scattering analysis and cytology of Mac-1⁺/Gr-1^int cells. (A) FSC histograms of Mac-1⁺/Gr-1^int (box) or Mac-1⁺/Gr-1^high (broken box) cells collected from FPs of groups of 10 BALB/c mice on day 3 after inoculation with RQ461 or RM461 infection (inoculum of 1 × 10⁶ PFUs). All histograms are from gated cell populations as indicated by boxed areas (i,ii,iii). (B) FSC histograms of MACS-enriched Mac-1⁺/Gr-1⁺ cells (boxed areas i and ii) from PB on day 3 or day 5 after inoculation with RQ461 (inoculum of 1 × 10⁶ PFUs). (C) FCM analysis of BM cells from uninfected mice analyzed by FCM for Mac-1 and Gr-1 after gating on FSC^low or FSC^high populations (boxed areas i and ii), indicating the percentage of total Mac-1⁺/Gr-1^int cells (× 600) in each population. (D-F) May-Grunwald-Giemsa-stained cells from FPs or PB of groups of 20 mice at 48 hours after inoculation (inoculum of 1 × 10⁶ PFUs), prepared as described for Figure 2. (D) FSC^low/Mac-1⁺/Gr-1^int cells from FPs. (E) FSC^high/Mac-1⁺/Gr-1^int cells from FPs. (F) Mac-1⁺/Gr-1^int cells from PB.

Figure 7.

FCM analysis of Mac-1⁺/Gr-1^int cells for CD31, Sca-1, and c-kit. FCM analysis of the FP infiltrates from groups of 10 infected BALB/c mice at 48 hours after inoculation with RQ461 (inoculum of 1 × 10⁶ PFUs) or mice at 4 hours after the fourth N-Met-MCK-2 injection. (A) Histograms of Mac-1⁺/Gr-1^int (solid box) or Mac-1⁺/Gr-1^high population (dotted box) FP cells from RQ461-infected mice, showing expression levels of CD31 (PECAM-1), Sca-1 (Ly-6A/E), and c-kit (CD117). (B) Histograms of Mac-1⁺/Gr-1^int (solid box) or Mac-1⁺/Gr-1^high (dotted box) FP cells from N-Met-MCK2-treated mice showing expression levels of CD31 (PECAM-1). Background staining with an isotype antibody control is indicated as a dashed line in each histogram.

Figure 8.

MCMV replication and dissemination in MCP-1^-/- CCR2^-/- mice. (A) Time-course analysis of FP swelling in 5 BALB/c (▪) and 5 MCP-1^-/- CCR2^-/- () mice following inoculation with 1 × 10⁶ PFUs of mck-expressing RM427⁺. The mean and standard deviation (error bars) are indicated. (B) Viremia levels determined by ICA on groups of 3 mice at day 5 after inoculation (intraperitoneal route) with 10⁵ PFUs of wild-type RQ461 in BALB/c and MCP-1^-/- CCR2^-/- mice. Mean (vertical line) and standard deviation (error bars) are indicated, along with data points from individual mice. (C) Titers of virus in spleen, lungs, and liver of RQ461 in BALB/c and MCP-1^-/- CCR2^-/- mice (same infection as panel A). (D) FCM analysis of Mac-1⁺/Gr-1^int myeloid cells recovered from FP of MCP-1^-/- CCR2^-/- mice. FP leukocytes were prepared from groups of 3 mice at day 5 after inoculation with mck-expressing RM427⁺ or mck-mutant RM461 lacZ-tagged viruses (inoculum of 1 × 10⁶ PFUs). FCM analysis and layout is described in the legend to Figure 2. (E) Day 14 virus titers in SGs following inoculation (FP route) with 10⁷ PFUs of RQ461 () or RM461 (□).