mune responses, but the Dendritic cells (DC) migrate from sites of inflammation to lymph nodes to initiate

rimaryimp 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. 
    　Introduction 
　　Pulmonary immune response initiation depends crucially on dendritic cells (DC),4 the potent APC essential to activate naive T cells (1, 2, 3). Within unperturbed lungs, immature DC (iDC) reside in airway epithelium, alveolar septae, and the connective tissue surrounding pulmonary vessels (4), but are rarely found within the alveolar spaces. iDC are specialized for Ag uptake and express only low surface amounts of MHC class II and costimulatory molecules. In response to inflammatory stimuli, DC increase expression of these molecules and become functional APC (5, 6). Simultaneously, DC up-regulate CCR7 and migrate to draining lymph nodes under the influence of the CCR7 ligands CCL19 and CCL21 (7, 8, 9). Migration of DC to lymph nodes is rapid, peaking within 24–48 h of Ag challenge (10, 11, 12, 13).

　　This well-described efflux of lung DC to the draining lymph node implies that peripheral tissues must become depleted of APC during inflammation, unless they are replaced. Although it is generally accepted that DC are replenished in lungs from peripheral blood precursors, direct evidence of such recruitment is limited (14, 15, 16, 17, 18, 19), and the potential molecular mechanisms controlling it are incompletely understood. Knowledge of such mechanisms could prove vital in the development of new therapeutics targeting immune-mediated lung diseases such as asthma and chronic obstructive pulmonary disease.

　　The goal of this study was to determine the kinetics with which DC are recruited to the interstitial and alveolar compartments of the lung during the response to lung inflammation, and to analyze the molecular requirements for their appearance. We used a model of CD4 T cell-dependent lung inflammation induced by a single intratracheal (i.t.) challenge of Ag-primed mice, using the particulate Ag SRBC (20). This model was chosen for its ability to induce an acute, self-limited inflammatory response with a clearly definable onset and well-established kinetics. Previous studies have proven this model system useful in defining the molecular mechanisms of lung leukocyte recruitment (21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33). Following i.t. Ag challenge, we assessed the presence of DC within the peripheral blood, the lung interstitium, and the alveolar spaces. We used gene-targeted mice to examine two classes of molecules that might mediate DC recruitment to the lungs. The first were the endothelial selectins, E-selectin (CD62E) and P-selectin (CD62P). In this model system, the endothelial selectins are markedly up-regulated (27) and are essential for recruitment of some lymphocyte subsets (30, 32). iDC bind E- and P-selectins under shear stress (15), making this adhesive system a plausible requirement for their recruitment. The second class of potential recruitment molecules were the chemokine receptors CCR2 and CCR6, which have recently been implicated in DC recruitment to the lung (19, 34, 35), tonsils (36, 37), and injured skin (36, 37, 38). Our results demonstrate that, in response to a single Ag challenge, the frequency of DC within peripheral blood increased rapidly, followed by a marked accumulation of DC within the lung interstitium and subsequently in the alveolar spaces. We further show that this process was independent of endothelial selectins, but that deficiency of chemokine receptor expression altered the efficacy with which DC were recruited into either the interstitial or alveolar compartments. 
    Materials and Methods 

　　Animals

　　Specific pathogen-free inbred female C57BL/6 mice purchased from Charles River Laboratories were used except as specified. Three strains of gene-targeted mice, each on a C57BL/6J background, were bred locally. Each strain has been described previously: E- and P-selectin double knockouts (E–P–) (39), chemokine receptor 2 gene knockouts (CCR2–/–) (40), and chemokine receptor 6 gene knockouts (CCR6–/–) (41). Mice were used in experiments at 8–14 wk of age. Mice were housed in the Animal Care Facility at the Ann Arbor Veterans Affairs Health System, which is fully accredited by the American Association for Accreditation of Laboratory Animal Care. Mice were provided standard animal chow and chlorinated tap water ad libitum. This study complied with the 1996 National Academy of Sciences Guide for the Care and Use of Laboratory Animals (www.nap.edu/readingroom/books/labrats/) and followed a protocol approved by the Animal Care Subcommittee of the local Institutional Review Board.