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-ß 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.

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

BACKGROUND: 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. PROCEDURE: Cross-sectional cohort study of children with SCA who participated in the Sleep Asthma Cohort Study. RESULTS: Fibrocyte levels were obtained from 45 children with SCA and 24 controls. Mean age of SCA cases was 14 years and 53% were female. In children with SCA, levels of circulating fibrocytes were greater than controls (P < 0.01). The fibrocytes expressed a hierarchy of chemokine receptors, with CXCR4 expressed on the majority of cells and CCR2 and CCR7 expressed on a smaller subset. Almost half of fibrocytes demonstrated α-smooth muscle actin activation. Increased fibrocyte levels were associated with a higher reticulocyte count (P = 0.03) and older age (P = 0.048) in children with SCA. However, children with increased levels of fibrocytes were not more likely to have asthma or lower percent predicted forced expiratory volume in 1 sec/forced vital capacity (FEV1 /FVC) or FEV1 than those with lower fibrocyte levels. CONCLUSIONS: Higher levels of fibrocytes in children with SCA compared to controls may be due to hemolysis. Longitudinal studies may be able to better assess the relationship between fibrocyte level and pulmonary dysfunction.

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.

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)-1alpha, MIP-1gamma, 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-1gamma in inflamed lungs. Eosinophil production of C10 and MIP-1gamma correlated with the marked influx of CD11b(high) 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.

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.

Molecular imaging of endothelial vascular cell adhesion molecule-1 expression and inflammatory cell recruitment during vasculogenesis and ischemia-mediated arteriogenesis.

BACKGROUND: Inflammatory responses contribute to vascular remodeling during tissue repair or ischemia. We hypothesized that inflammatory cell recruitment and endothelial cell activation during vasculogenesis and ischemia-mediated arteriogenesis could be temporally assessed by noninvasive molecular imaging. METHODS AND RESULTS: Contrast ultrasound perfusion imaging and molecular imaging with microbubbles targeted to activated neutrophils, alpha(5)-integrins, or vascular cell adhesion molecule (VCAM-1) were performed in murine models of vasculogenesis (subcutaneous matrigel) or hind-limb ischemia produced by arterial occlusion in wild-type or monocyte chemotactic protein-1-deficient mice. In subcutaneous matrigel plugs, perfusion advanced centripetally between days 3 and 10. On targeted imaging, signal enhancement from alpha(5)-integrins and VCAM-1 coincided with the earliest appearance of regional blood flow. Targeted imaging correlated temporally with histological evidence of channel formation by alpha(5)-integrin-positive monocytes, followed by the appearance of spindle-shaped cells lining the channels that expressed VCAM-1. In ischemic hind-limb tissue, skeletal muscle blood flow and arteriolar density increased progressively between days 2 and 21 after arterial ligation. Targeted imaging demonstrated early signal enhancement for neutrophils, monocyte alpha(5)-integrin, and VCAM-1 at day 2 when blood flow was very low (<20% control). The neutrophil signal declined precipitously between days 2 and 4, whereas VCAM-1 and monocyte signal persisted to day 7. In mice deficient for monocyte chemotactic protein-1, monocyte-targeted signal was severely reduced compared with wild-type mice (1.2+/-0.6 versus 10.5+/-8.8 video intensity units on day 4; P<0.05), although flow responses were only mildly impaired. CONCLUSIONS: Different components of the inflammatory response that participate in vascular development and remodeling can be assessed separately with targeted molecular imaging.

Diverse and potent chemokine production by lung CD11bhigh dendritic cells in homeostasis and in allergic lung inflammation.

Lung CD11c(high) dendritic cells (DC) are comprised of two major phenotypically distinct populations, the CD11b(high) DC and the integrin alpha(E)beta(7)(+) 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 CD11c(high) DC types in chemokine mRNA expression were found. CD11b(high) 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 CD11c(high) lung cells including macrophages, and ELISA determination of sort-purified CD11c(high) cell culture supernatants, further showed that CD11b(high) DC produced the highest levels of 9 of 14 and 5 of 7 chemokines studied, respectively. Upon LPS stimulation in vitro and in vivo, CD11b(high) DC remained the highest producer of 7 of 10 of the most highly produced chemokines. Induction of airway hyperreactivity and lung inflammation increased lung CD11b(high) 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 CD11b(high) DC is one of the most important leukocyte types in chemokine production and it is readily distinguishable from CD103(+) DC in this secretory function.

Enhanced allergen-induced airway inflammation in paucity of lymph node T cell (plt) mutant mice.

BACKGROUND: Dendritic cells and lymphocytes play a central role in allergic asthma. Chemokines for these cells include the CCR7 agonists secondary lymphoid chemokine/CCL21 and EBV-induced lymphoid chemokine/CCL19, but their role in allergic asthma is poorly understood. OBJECTIVE: We sought to determine the effects of abrogation of lymphoid tissue expression of CCR7 agonists on allergic airway responses. METHODS: Paucity of lymphocyte T cell (plt) mutant mice, deficient in EBV-induced lymphoid chemokine/CCL19 and the lymphoid form of secondary lymphoid chemokine/CCL21, were evaluated in an established ovalbumin (OVA)-induced asthma model (plt-OVA group) and compared with similarly immunized +/+ BALB/c mice (+/+OVA group). RESULTS: APTI responses to methacholine increased similarly in OVA-challenged plt and +/+ mice. However, airway inflammation was strikingly enhanced in plt-OVA mutants over +/+OVA mice and included increased numbers of eosinophils, CD4 and B cells, neutrophils, and total leukocytes in bronchoalveolar lavage fluid and inflammatory cell cuffing around pulmonary arterioles. Enhanced airway inflammation was accompanied by an increase in lung T(H)2 activity, with increased levels of IL-4 and monocyte-derived chemoattractant/CCL22. CONCLUSIONS: Induction of allergic asthma in mutant mice with impaired CCR7 responses results in characteristics that resemble severe asthma in human subjects, including severe bronchial lymphocytosis, eosinophilia, and neutrophilia, but not in enhancement in airway hyperreactivity. CLINICAL IMPLICATIONS: Disruption of chemokines responsible for trafficking of antigen-processing cells and lymphocytes to the draining lymph nodes might lead to enhanced allergic airway responses.

ICAM-1 and LFA-1 play critical roles in LPS-induced neutrophil recruitment into the alveolar space.

Neutrophil recruitment into lung constitutes a major response to airborne endotoxins. In many tissues endothelial intercellular adhesion molecule-1 (ICAM-1) interacts with lymphocyte function associated antigen-1 (LFA-1) on neutrophils, and this interaction plays a critical role in neutrophil recruitment. There are conflicting reports about the role of ICAM-1 in neutrophil recruitment into lungs. We studied neutrophil recruitment into alveolar space in a murine model of aerosolized LPS-induced lung inflammation. LPS induces at least a 100-fold increase in neutrophil numbers in alveolar space, as determined by flow cytometry of bronchoalveolar lavage fluid. Neutrophil recruitment was reduced by 54% in ICAM-1 null mice and by 45% in LFA-1 null mice. In wild-type mice treated with anti-ICAM-1 and anti-LFA-1 antibodies, there was 51 and 58% reduction in the neutrophil recruitment, respectively. In chimeric mice, generated by the transplantation of mixtures of bone marrows from LFA-1 null and wild-type mice, the normalized recruitment of LFA-1 null neutrophils was reduced by 60% compared with wild-type neutrophils. Neither the treatment of ICAM-1 null mice with a function-blocking antibody to LFA-1 nor the treatment of LFA-1 null mice with anti-ICAM-1 antibody resulted in further reduction in the recruitment compared with untreated ICAM-1 null and LFA-1 null mice. We conclude that ICAM-1 and LFA-1 play critical roles in the recruitment of neutrophils into the alveolar space in aerosolized LPS-induced lung inflammation, and LFA-1 serves as a ligand of ICAM-1 in the lung.

A major lung CD103 (alphaE)-beta7 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 alpha(E) (CD103) beta(7)-positive and I-A(high)CD11c(high)-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 alpha(E)beta(7)(+) DC (alphaE-DC) were distinct from intraepithelial lymphocytes and distinguishable from CD11b(high) 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, alphaE-DC numbers were increased in lungs, and their costimulation and adhesion molecules were up-regulated. These studies show that alphaE-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.

Reversible ST-segment elevation associated with atelectasis of the left lung.

A 22-year-old male with cerebral palsy and respiratory failure had acute reversible ST-segment elevation in the inferior leads during acute collapse of the left lung, which resolved with re-expansion of the left lung several hours later. This suggests that major lung actelectasis needs to be added to the group of noncardiac conditions considered when evaluating ST elevation in the critically ill patient.

Comparison of diagnostic accuracies in outpatients and hospitalized patients of D-dimer testing for the evaluation of suspected pulmonary embolism.

BACKGROUND: The ability of various D-dimer assays to exclude the diagnosis of thromboembolic diseases is controversial. We examined the diagnostic accuracy of two D-dimer methods in hospitalized patients and outpatients. METHODS: We studied consecutive patients for whom D-dimer testing was ordered for investigation of suspected pulmonary embolism. We measured D-dimer by an ELISA (VIDAS D-dimer) and an enhanced microlatex immunoassay method (Diagnostica Stago STA Liatest D-di). Patient diagnoses were based on imaging studies or, when these were not performed, on follow-up by review of medical records 3 months later. RESULTS: We examined 233 hospitalized patients and 234 outpatients with a mean age of 58 years (range, 1-92 years) and a female-to-male ratio of 1.4 to 1. Thromboembolism was present in 8% of outpatients and 12% of hospitalized patients. In outpatients, the negative predictive values were 98% [95% confidence interval (CI), 93-100%] and 99% (94-100%) for the microlatex and ELISA methods, respectively, at the recommended cutoffs. Areas under the ROC curves were similar for the two methods [0.77 (95% CI, 0.67-0.87) and 0.81 (0.73-0.89), respectively]. By contrast, in hospitalized patients, the confidence intervals for the areas under the ROC curves included 0.5 [0.60 (95% CI, 0.50-0.71) and 0.56 (0.44-0.67)]. CONCLUSIONS: For hospitalized patients, in contrast to outpatients, the diagnostic accuracy of D-dimer testing for pulmonary embolism is poor in a tertiary care setting, presumably reflecting thrombosis and comorbidities, other than pulmonary embolism, that increase the D-dimer concentrations in these patients. The patient population studied appears more important than assay method in studies of the diagnostic accuracy of D-dimer testing.

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-beta (TGF-beta) 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.