C. Edward Rose

A Major Lung CD103 (αE)-β7 Integrin-Positive Epithelial Dendritic Cell Population Expressing Langerin and Tight Junction Proteins

Dendritic cells (DC) mediate airway Ag presentation and play key roles in asthma and infections. Although DC subsets are known to perform different functions, their occurrence in mouse lungs has not been clearly defined. In this study, three major lung DC populations have been found. Two of them are the myeloid and plasmacytoid DC (PDC) well-characterized in other lymphoid organs. The third and largest DC population is the integrin αE (CD103) β7-positive and I-AhighCD11chigh-DC population. This population was found to reside in the lung mucosa and the vascular wall, express a wide variety of adhesion and costimulation molecules, endocytose avidly, present Ag efficiently, and produce IL-12. Integrin αEβ7+ DC (αE-DC) were distinct from intraepithelial lymphocytes and distinguishable from CD11bhigh myeloid and mPDCA-1+B220+Gr-1+ PDC populations in surface marker phenotype, cellular functions, and tissue localization. Importantly, this epithelial DC population expressed high levels of the Langerhans cell marker Langerin and the tight junction proteins Claudin-1, Claudin-7, and ZO-2. In mice with induced airway hyperresponsiveness and eosinophilia, αE-DC numbers were increased in lungs, and their costimulation and adhesion molecules were up-regulated. These studies show that αE-DC is a major and distinct lung DC population and a prime candidate APC with the requisite surface proteins for migrating across the airway epithelia for Ag and pathogen capture, transport, and presentation. They exhibit an activated phenotype in allergen-induced lung inflammation and may play significant roles in asthma pathogenesis.

The chemokine receptors CCR2 and CX3CR1 mediate monocyte/macrophage trafficking in kidney ischemia–reperfusion injury

Chemokines and their receptors such as CCR2 and CX3CR1 mediate leukocyte adhesion and migration into injured tissue. To further define mechanisms of monocyte trafficking during kidney injury we identified two groups of F4/80-positive cells (F4/80(low) and F4/80(high)) in the normal mouse kidney that phenotypically correspond to macrophages and dendritic cells, respectively. Following ischemia and 3 h of reperfusion, there was a large influx of F4/80(low) inflamed monocytes, but not dendritic cells, into the kidney. These monocytes produced TNF-alpha, IL-6, IL-1alpha and IL-12. Ischemic injury induced in CCR2(-/-) mice or in CCR2(+/+) mice, made chimeric with CCR2(-/-) bone marrow, resulted in lower plasma creatinine levels and their kidneys had fewer infiltrated F4/80(low) macrophages compared to control mice. CX3CR1 expression contributed to monocyte recruitment into inflamed kidneys, as ischemic injury in CX3CR1(-/-) mice was reduced, with fewer F4/80(low) macrophages than controls. Monocytes transferred from CCR2(+/+) or CX3CR1(+/-) mice migrated into reperfused kidneys better than monocytes from either CCR2(-/-) or CX3CR1(-/-) mice. Adoptive transfer of monocytes from CCR2(+/+) mice, but not CCR2(-/-) mice, reversed the protective effect in CCR2(-/-) mice following ischemia-reperfusion. Egress of CD11b(+)Ly6C(high) monocytes from blood into inflamed kidneys was CCR2- and CX3CR1-dependent. Our study shows that inflamed monocyte migration, through CCR2- and CX3CR1-dependent mechanisms, plays a critical role in kidney injury following ischemia reperfusion.

Diverse and Potent Chemokine Production by Lung CD11bhigh Dendritic Cells in Homeostasis and in Allergic Lung Inflammation

Lung CD11chigh dendritic cells (DC) are comprised of two major phenotypically distinct populations, the CD11bhigh DC and the integrin αEβ7+ DC (CD103+ DC). To examine whether they are functionally distinguishable, global microarray studies and real-time PCR analysis were performed. Significant differences between the two major CD11chigh DC types in chemokine mRNA expression were found. CD11bhigh DC is a major secretory cell type and highly expressed at least 16 chemokine mRNA in the homeostatic state, whereas CD103+ DC highly expressed only 6. Intracellular chemokine staining of CD11chigh lung cells including macrophages, and ELISA determination of sort-purified CD11chigh cell culture supernatants, further showed that CD11bhigh DC produced the highest levels of 9 of 14 and 5 of 7 chemokines studied, respectively. Upon LPS stimulation in vitro and in vivo, CD11bhigh DC remained the highest producer of 7 of 10 of the most highly produced chemokines. Induction of airway hyperreactivity and lung inflammation increased lung CD11bhigh DC numbers markedly, and they produced comparable or higher amounts of 11 of 12 major chemokines when compared with macrophages. Although not a major producer, CD103+ DC produced the highest amounts of the Th2-stimulating chemokines CCL17/thymus and activation-related chemokine and CCL22/monocyte-derived chemokine in both homeostasis and inflammation. Significantly, CCL22/monocyte-derived chemokine exhibited regulatory effects on CD4+ T cell proliferation. Further functional analysis showed that both DC types induced comparable Th subset development. These studies showed that lung CD11bhigh DC is one of the most important leukocyte types in chemokine production and it is readily distinguishable from CD103+ DC in this secretory function.

Significant Involvement of CCL2 (MCP-1) in Inflammatory Disorders of the Lung

Mounting evidence suggests that CCL2 (MCP‐1) and its hematopoietic cell receptor CC chemokine receptor 2 (CCR2) are involved in inflammatory disorders of the lung. In animal models of allergic asthma, idiopathic pulmonary fibrosis (IPF), and bronchiolitis obliterans syndrome (BOS), CCL2 expression and protein production are increased and the disease process is attenuated by CCL2 immunoneutralization. Mechanisms by which CCL2 may be acting include recruitment of regulatory and effector leukocytes; stimulation of histamine or leukotriene release from mast cells or basophils; induction of fibroblast production of transforming growth factor‐β (TGF‐β) and procollagen; and enhancement of Th2 polarization. Recently, polymorphism for CCL2 has been described with increased cytokine‐induced release of CCL2 by monocytes and increased risk of allergic asthma. These studies identify potentially important roles for CCL2 in these lung inflammatory disorders. While CCL2 inhibition in patients with acute respiratory distress syndrome (ARDS) may be hazardous by interfering with defense against bacteremia, future studies are needed to determine if CCL2/CCR2 antagonism will offer breakthrough therapy for patients with allergic asthma, IPF, or BOS, and to confirm the hypothesis that CCL2 polymorphism places patients at greater risk for these disorders.

Enhanced airway Th2 response after allergen challenge in mice deficient in CC chemokine receptor-2 (CCR2).

To evaluate the role of CCR2 in allergic asthma, mutant mice deficient in CCR2 (CCR2−/−) and intact mice were sensitized with i.p. OVA with alum on days 0 and 7, and challenged by inhalation with nebulization of either OVA or saline. Airway hyperreactivity, measured by the methacholine-provoked increase in enhanced pause, was significantly increased (p < 0.05) in OVA-challenged CCR2−/− mutant mice, compared with comparably challenged CCR2+/+ mice. OVA-challenged CCR2−/− mutants also were also found to have enhanced bronchoalveolar lavage fluid eosinophilia, peribronchiolar cellular cuffing, and Ig subclass switching, with increase in OVA-specific IgG1 and IgE. In addition, RNase protection assay revealed increased whole lung expression of IL-13 in OVA-challenged CCR2−/− mutants. Unexpectedly, serum monocyte chemotactic protein-1 levels were 8-fold higher in CCR2−/− mutants than in CCR2+/+ mice sensitized to OVA, but OVA challenge had no additional effect on circulating monocyte chemotactic protein-1 in either genotype. Ag stimulation of lymphocytes isolated from OVA-sensitized CCR2 mutants revealed a significant increase (p < 0.05) in IL-5 production, which differed from OVA-stimulated lymphocytes from sensitized CCR2+/+ mice. These experiments demonstrate an enhanced response in airway reactivity and in lung inflammation in CCR2−/− mutant mice compared with comparably sensitized and challenged CCR2+/+ mice. These observations suggest that CC chemokines and their receptors are involved in immunomodulation of atopic asthma.

Intratracheal Priming with Ovalbumin- and Ovalbumin 323–339 Peptide-Pulsed Dendritic Cells Induces Airway Hyperresponsiveness, Lung Eosinophilia, Goblet Cell Hyperplasia, and Inflammation

Dendritic cells (DC) are the primary APC responsible for the capture of allergens in the airways and the shuttling of processed allergens to the draining lymph nodes where Ag presentation and T cell activation take place. The mechanism of this Ag handling and presentation in asthma is poorly understood. In addition, the feasibility of asthma induction by DC priming has not been directly tested. In this report an asthma model using intratracheally (i.t.) injected splenic DC was used to address these issues. DC pulsed with a model Ag OVA or the major MHC class II-restricted OVA T epitope peptide OVA323–339 and instilled i.t. primed mice to exhibit asthma-like diseases. With OVA as the Ag, mice exhibit airway hyperresponsiveness (AHR), lung eosinophilia and inflammation, and pulmonary goblet cell hyperplasia. In OVA323–339-immunized mice, AHR and goblet cell hyperplasia were noted, with little eosinophilia and parenchymal inflammation. The latter finding provides evidence for dissociating AHR from eosinophilia. In both cases mediastinal node hypertrophy occurred, and high levels of Th2 cytokines were produced by the lung and mediastinal lymph node cells (LNC). Interestingly, mediastinal LNC also produced high levels of Th1 cytokines. Lung cells produced much less Th1 cytokines than these LNC. These results demonstrate that DC when introduced i.t. are potent in inducing asthma-like diseases by recruiting lymphocytes to the lung-draining lymph nodes and by stimulating Th2 responses and also suggest that the lung environment strongly biases T cell responses to Th2.

Murine lung eosinophil activation and chemokine production in allergic airway inflammation

Eosinophils play important roles in asthma and lung infections. Murine models are widely used for assessing the functional significance and mechanistic basis for eosinophil involvements in these diseases. However, little is known about tissue eosinophils in homeostasis. In addition, little data on eosinophil chemokine production during allergic airway inflammation are available. In this study, the properties and functions of homeostatic and activated eosinophils were compared. Eosinophils from normal tissues expressed costimulation and adhesion molecules B7-1, B7-2 and ICAM-1 for Ag presentation but little major histocompatibility complex (MHC) class II, and were found to be poor stimulators of T-cell proliferation. However, these eosinophils expressed high levels of chemokine mRNA including C10, macrophage inflammatory protein (MIP)-1α, MIP-1γ, MIP-2, eotaxin and monocyte chemoattractant protein-5 (MCP-5), and produced chemokine proteins. Eosinophil intracellular chemokines decreased rapidly with concomitant surface marker downregulation upon in vitro culturing consistent with piecemeal degranulation. Lung eosinophils from mice with induced allergic airway inflammation exhibited increased chemokines mRNA expression and chemokines protein production and upregulated MHC class II and CD11c expression. They were also found to be the predominant producers of the CCR1 ligands CCL6/C10 and CCL9/MIP-1γ in inflamed lungs. Eosinophil production of C10 and MIP-1γ correlated with the marked influx of CD11bhigh lung dendritic cells during allergic airway inflammation and the high expression of CCR1 on these dendritic cells (DCs). The study provided baseline information on tissue eosinophils, documented the upregulation of activation markers and chemokine production in activated eosinophils, and indicated that eosinophils were a key chemokine-producing cell type in allergic lung inflammation.

Number, activation, and differentiation of circulating fibrocytes correlate with asthma severity

BACKGROUND A biomarker that predicts poor asthma control would be clinically useful. Fibrocytes are bone marrow-derived circulating progenitor cells that have been implicated in tissue fibrosis and T(H)2 responses in asthmatic patients. OBJECTIVE We sought to test the hypothesis that the concentration and activation state of peripheral blood fibrocytes correlates with asthma severity. METHODS By using fluorescence-activated cell sorting analysis, fibrocytes (CD45(+) and collagen 1 [Col1](+)) were enumerated and characterized in the buffy coats of fresh peripheral blood samples from 15 control subjects and 40 asthmatic patients. RESULTS Concentrations of peripheral blood total (CD45(+)Col1(+)), activated (the TGF-β transducing protein phosphorylated SMAD2/3 [p-SMAD2/3](+) or phosphorylated AKT [p-AKT](+)), and differentiated (α-smooth muscle actin [α-SMA](+)) fibrocytes were increased in asthmatic patients compared with control subjects. The increase in total and CD45(+)Col1(+)CXCR4(+) fibrocytes was primarily seen in patients with severe asthma (Global Initiative for Asthma steps 4-5) as opposed to those with milder asthma (Global Initiative for Asthma steps 1-3). In addition, numbers of circulating α-SMA(+) and α-SMA(+)CXCR4(+) fibrocytes were increased in asthmatic patients experiencing an asthma exacerbation in the preceding 12 months. A significant correlation (P < .05) was observed between CD45(+)Col1(+)CXCR4(+) fibrocytes and the activation phenotypes CD45(+)Col1(+)p-SMAD2/3(+) and CD45(+)Col1(+)p-AKT(+). CONCLUSION There was correlation between circulating fibrocyte subsets and asthma severity, and there was an increased number of activated/differentiated fibrocytes in circulating blood of asthmatic patients experiencing an exacerbation in the preceding 12 months.

The Role of Fibrocytes in Sickle Cell Lung Disease

Background Interstitial lung disease is a frequent complication in sickle cell disease and is characterized by vascular remodeling and interstitial fibrosis. Bone marrow-derived fibrocytes have been shown to contribute to the pathogenesis of other interstitial lung diseases. The goal of this study was to define the contribution of fibrocytes to the pathogenesis of sickle cell lung disease. Methodology/Principal Findings Fibrocytes were quantified and characterized in subjects with sickle cell disease or healthy controls, and in a model of sickle cell disease, the NY1DD mouse. The role of the chemokine ligand CXCL12 in trafficking of fibrocytes and phenotype of lung disease was examined in the animal model. We found elevated concentration of activated fibrocytes in the peripheral blood of subjects with sickle cell disease, which increased further during vaso-occlusive crises. There was a similar elevations in the numbers and activation phenotype of fibrocytes in the bone marrow, blood, and lungs of the NY1DD mouse, both at baseline and under conditions of hypoxia/re-oxygenation. In both subjects with sickle cell disease and the mouse model, fibrocytes expressed a hierarchy of chemokine receptors, with CXCR4 expressed on most fibrocytes, and CCR2 and CCR7 expressed on a smaller subset of cells. Depletion of the CXCR4 ligand, CXCL12, in the mouse model resulted in a marked reduction of fibrocyte trafficking into the lungs, reduced lung collagen content and improved lung compliance and histology. Conclusions These data support the notion that activated fibrocytes play a significant role in the pathogenesis of sickle cell lung disease.

Increased circulating fibrocytes are associated with higher reticulocyte percent in children with sickle cell anemia

Interstitial lung disease is common in patients with sickle cell anemia (SCA). Fibrocytes are circulating cells implicated in the pathogenesis of pulmonary fibrosis and airway remodeling in asthma. In this study, we tested the hypotheses that fibrocyte levels are: (1) increased in children with SCA compared to healthy controls, and (2) associated with pulmonary disease.