CCR2 and CCR6, but Not Endothelial Selectins, Mediate the Accumulation of Immature Dendritic Cells within the Lungs of Mice in Response to Particulate Antigen

Abstract

Dendritic cells (DC) migrate from sites of inflammation to lymph nodes to initiate primary immune responses, but the molecular mechanisms by which DC are replenished in the lungs during ongoing pulmonary inflammation are unknown. To address this question, we analyzed the secondary pulmonary immune response of Ag-primed mice to intratracheal challenge with the particulate T cell-dependent Ag sheep erythrocytes (SRBC). We studied wild-type C57BL/6 mice and syngeneic gene-targeted mice lacking either both endothelial selectins (CD62E and CD62P), or the chemokine receptors CCR2 or CCR6. DC, defined as non-autofluorescent, MHC class II+CD11cmod cells, were detected in blood, enzyme-digested minced lung, and bronchoalveolar lavage fluid using flow cytometry and immunohistology. Compared with control mice, Ag challenge increased the frequency and absolute numbers of DC, peaking at day 1 in peripheral blood (6.5-fold increase in frequency), day 3 in lung mince (20-fold increase in total DC), and day 4 in bronchoalveolar lavage fluid (55-fold increase in total DC). Most lung DC expressed CD11c, CD11b, and low levels of MHC class II, CD40, CD80, and CD86, consistent with an immature myeloid phenotype. DC accumulation depended in part upon CCR2 and CCR6, but not endothelial selectins. Thus, during lung inflammation, immature myeloid DC from the bloodstream replace emigrating immature DC and transiently increase total intrapulmonary APC numbers. Early DC recruitment depends in part on CCR2 to traverse vascular endothelium, plus CCR6 to traverse alveolar epithelium. The recruitment of circulating immature DC represents a potential therapeutic step at which to modulate immunological lung diseases.

Chemokine Receptor 2-Mediated Accumulation of Fungicidal Exudate Macrophages in Mice That Clear Cryptococcal Lung Infection

“…Our use of AF to distinguish macrophages (AF ϩ ) from lung DCs (AF Ϫ ) represents a critical step in our identification process because both cell types express CD11c. This distinction, well described in two prior studies 41,45 and in other infectious and noninfectious models of pulmonary inflammation, 42,[51][52][53]68 permits more specific identification and enumeration of macrophage and DC populations in the lungs of mice. In our model, AF ϩ macrophages, purified by cell sorting, are large cells (with high forward and side scatter) displaying numerous intracytoplasmic vacuoles, some containing cryptococci, consistent with macrophage morphological features (Figure 1).…”

“…41 The peripheral blood mononuclear cells were isolated from peripheral blood obtained from the retro-orbital vein of deeply anesthetized mice and processed as previously described. 42 Spleens were excised and leukocytes were dispersed by mincing and mechanical disruption. Bone marrow was harvested by removing the ends from both femurs with a number 10 blade scalpel (after removal of skin and muscle) and flushing the marrow with 2 ml of PBS, using a 3-ml syringe and a 25½-gauge needle, into a sterile 100 ϫ 15-mm dish; leukocytes were dispersed further by mechanical disruption.…”

“…Next, we set consecutive gates which identified CD11c ϩ cells ( Figure 1A, left dot plot) and distinguished autofluorescent (AF ϩ ) macrophages from non-AF (AF Ϫ ) dendritic cells (DCs) ( Figure 1A, middle dot plot), as previously described. 41,45,[51][52][53] Last, we used relative expression of CD11b to distinguish CD11b-negative AMs (CD11c ϩ AF ϩ CD11b Ϫ ) 42,54,55 and CD11b-positive ExMs (CD11c ϩ AF ϩ CD11b ϩ ) 33,35 ( Figure 1A, right dot plot; gates labeled AM and ExM). Proper gate placement for AF and expression of CD11c and CD11b were determined using AMs obtained from bronchoalveolar lavage fluid of uninfected mice (which consists of Ͼ90% CD11c ϩ AF ϩ CD11b Ϫ AMs; data not shown).…”

Chemokine Receptor CCR2 but Not CCR5 or CCR6 Mediates the Increase in Pulmonary Dendritic Cells during Allergic Airway Inflammation

“…Experiments in CCR6 Ϫ/Ϫ mice showed a reduced inflammatory response that was partially attributed to decreased pulmonary DC numbers (14). Another study reported a diminished early accumulation of lung DCs in challenged CCR2 Ϫ/Ϫ and CCR6 Ϫ/Ϫ mice (15). Activation and maturation of DCs was severely compromised in CCR2 Ϫ/Ϫ mice (16).…”

Urban particulate matter stimulation of human dendritic cells enhances priming of naive CD8 T lymphocytes

“…Induction of CCR7 on human mDCs by UPM, as we report, supports the results from murine experiments that diesel exhaust particle‐laden DCs translocate to lymph nodes after diesel exhaust particle instillation in to the lungs,31 and is important given that lymph nodes are the primary site for priming of new immune responses in vivo . Decreased expression of CCR2 and CCR6 on mDCs after exposure to UPM in the lungs as indicated by our results may act to release these mDCs from the pulmonary environment, as both are important in chemotaxis of inflammatory DCs in to the lungs 32, 33, 34. However, the resulting effects of down‐regulation of CCR2 and CCR6 on DCs are probably more complex – for example Sato and colleagues have previously shown CCR2 to have a role in migration of Langerhans cells (skin‐resident dendritic cells) to and within lymph nodes, and CCR2 −/− knockout mice to have an exaggerated T helper type 2 immune response to Leishmania major infection 35…”