Impaired lung dendritic cell activation in CCR2 knockout mice

Abstract

Dendritic cell (DC) recruitment is a hallmark event in antigen (Ag)-challenged lungs. We previously reported models for analyzing DC migration and activation in the lung after Th1- or Th2-eliciting pathogen Ag-bead challenge. To determine the role of chemokines in DC mobilization, we applied this analysis to CCR1, CCR2, CCR5, and CCR6 chemokine receptor knockout mice. Both Mycobacteria bovis protein Ags and helminthic, Schistosoma mansoni egg Ags elicited multiple chemokines, including CCR1, CCR2, CCR5, and to a lesser extent CCR6 ligands. DCs from wild-type lungs expressed transcripts for chemokine receptors, CCR1, CCR2, CCR5, and CXCR4. In all knockout strains, CD11c+ cells were recruited to Ag-beads likely because of receptor redundancy. However, DCs in CCR2-/- mice had significantly decreased MHCII and CD40 expression. This was associated with abrogated cytokine production in draining lymph node cultures. Analysis of local innate inflammation revealed a 50% reduction in macrophage recruitment in CCR2-/- mice. Bone marrow chimeras of mixed CCR2+/+ green fluorescent protein transgenic and CCR2-/- green fluorescent protein-negative cells confirmed the DC maturation defect was only among the latter population. In conclusion, CCR2 knockout confers an intrinsic DC activation defect and CCR2 ligands likely promote the local activation/maturation of inflammatory DCs.

Figure 1-4238

Chemokine receptor transcript expression among CD11c+ populations purified from naïve lungs, challenged lungs, and DLNs. CD11+ DCs were freshly isolated from naïve CBA/J mouse lungs (lung) or lungs (bead) and corresponding DLNs (node) from day 3 Ag-bead (SEA or PPD)-challenged CBA/J mice. Transcript expression was measured using real-time PCR. Bars are mean arbitrary units ± SD derived from three independent samples, in which each sample was generated from DCs isolated from a pool of five mice.

Figure 2-4238

Time course and induction of chemokine transcripts in lungs with innate Ag-bead granuloma formation. Lung tissues were collected from naïve or day 1, 2, or 3 Ag-bead-challenged CBA/J mice. Transcript expression was measured using real-time PCR. Bars indicated fold increases over naïve unchallenged lungs and were derived from lungs of five individual mice.

Figure 3-4238

Innate granuloma formation in chemokine receptor knockout mice. Day 3 Ag-bead granulomas were elicited in the various knockout strains as described in Materials and Methods. Lungs were then removed, prepared for histological sectioning, and subjected to morphometric analysis to determine average granuloma cross-sectional area. A minimum of 20 lesions was measured per mouse. Bars are means ± SEM. The dashed line indicates area occupied by the beads. Bars are grouped according to the background strain and are shown with the appropriate control strain (n = five to six mice per group). *, P < 0.05.

Figure 4-4238

Immunohistochemical and morphological detection of CD11c+ cells in Ag-bead granulomas elicited in control and chemokine receptor knockout strains. Frozen sections were obtained from day 3 bead-challenged lungs and stained for CD11c. Note red-brown staining cells within innate granulomas. CD11c+ cells were detectable in all knockout strains although overall cellularity was less in CCR2−/− mice. Wild-type B6X129 and CCR6 knockout mice (not shown) were similar to wild-type B6 controls (shown). Original magnifications, ×200.

Figure 5-4238

Flow cytometric scattergram analysis of CD11c+ populations in naïve and challenged control and CCR2−/− mice. The lungs of naïve or day 3 Ag-bead-challenged CCR2+/+ controls or CCR2−/− mice were enzymatically dispersed as described in Materials and Methods. The single cell suspensions from the lungs were then labeled with indicated antibodies and subjected to flow cytometric analysis. Note, the up-regulation of MHCII and CD40 observed in Ag-bead-challenged CCR2+/+ lungs failed to occur in the challenged lungs of CCR2−/− mice (top right quadrants). Percentages of total cells are shown in right quadrants.

Figure 6-4238

Innate granuloma formation in CCR2−/− and GFP transgenic bone marrow chimeric mice. Recipient CCR2+/+ wild-type or CCR2−/− mice were irradiated to ablate bone marrow and then reconstituted with CCR2−/−, GFP transgenic wild-type (*, CCR2+/+), or a 1:1 mixture of these. All recipients received a total of 10 million bone marrow cells. Three weeks after transfer, day 3 PPD-bead granulomas were elicited as described in Materials and Methods. Granulomatous lungs were then removed, prepared for histological sectioning, and subjected to morphometric analysis to determine average granuloma cross-sectional area. A minimum of 20 lesions was measured per mouse. The dashed line indicates area occupied by the beads. Bars are means ± SEM of three to four mice per group. *, P < 0.05.

Figure 7-4238

CCR2−/− DCs display a persistent maturation defect after co-transfer of CCR2+/+ and CCR2−/− bone marrow. Recipient CCR2−/− mice were irradiated to ablate bone marrow and then reconstituted with 1:1 mixture of CCR2−/−, and CCR2+/+ GFP transgenic wild-type. Recipients received a total of 10 million bone marrow cells. Three weeks after transfer, day 3 PPD-bead granulomas were elicited as described in Materials and Methods. Granulomatous lungs were removed, enzymatically dispersed, and subjected to flow cytometric analysis. The mix of CCR2+/+ and CCR2−/− cells was analyzed independently by gating on either GFP+ or GFP− cells. Note in the top right corner of scattergrams the population shift from MHCII^hi and CD40^hi to low expression in CCR2−/− mice. The dense population in bottom left corner of CCR2−/− mice represents CD45-GFP− cells derived from the bone marrow recipient. Scattergrams from a representative mouse are shown.