Alternatively activated macrophages (AAM) play a crucial role in type 2 immunity. Mice deficient in ST2, a receptor for the latest member of the IL-1 family, IL-33, have impaired type 2 immune responses. We therefore reasoned that IL-33/ST2 signaling may be involved in the differentiation and activation of AAM during airway inflammation. We report here that IL-33 changed the quiescent phenotype of alveolar macrophages toward an AAM phenotype that expressed mannose receptor, IL-4Ralpha, and produced high levels of CCL24 and CCL17 in an IL-13-dependent manner during IL-33-induced airway inflammation. Neutralization of AAM-derived CCL24 led to an amelioration of IL-33-induced eosinophilia in the lungs. Moreover, depletion of alveolar macrophages reduced IL-33-induced airway inflammation. Additionally, the attenuated OVA-induced airway inflammation in ST2(-/-) mice was associated with a decrease in AAM differentiation. In vitro, IL-33 amplified IL-13-induced polarization of alveolar- and bone marrow-derived macrophage toward an AAM phenotype by increasing the expression of arginase I, Ym1, as well as the production of CCL24 and CCL17. IL-13/IL-4Ralpha signaling was crucial for IL-33-driven AAM amplification by inducing the expression of ST2L. Finally, we showed that IL-33 was more abundantly expressed in the lung epithelial cells of asthma patients than those from healthy controls, suggesting that IL-33 may be involved in lung macrophage activation in clinical asthma. Taken together, we demonstrate here that IL-33/ST2 plays a significant role in the amplification of AAM polarization and chemokine production which contribute to innate and Ag-induced airway inflammation.

IL-33 has emerged as an important mediator in the immunopathogenesis of allergy and asthma. However, the role of IL-33 in eosinophil-mediated inflammation has not been fully explored. In this article, we report that IL-33 directly stimulates eosinophil differentiation from CD117(+) progenitors in an IL-5-dependent manner. Although resting eosinophils expressed moderate levels of the IL-33R alpha-chain (ST2L), eosinophils that accumulated in the airways of mice with OVA-induced asthma expressed increased amounts of ST2L. In vitro, IL-33 and GM-CSF are potent inducers of ST2L expression on eosinophils, and IL-33 induced the production of IL-13, CCL17, and TGF-beta by eosinophils. In adoptive-transfer experiments, IL-33 exacerbated eosinophil-mediated airway inflammation by increasing the levels of eosinophils, macrophages, lymphocytes, IL-13, TGF-beta, CCL3, CCL17, and CCL24 in the lungs. IL-33 also enhanced the eosinophil-mediated differentiation of airway macrophages toward the alternatively activated macrophage phenotype in an IL-13-dependent manner. Taken together, this study demonstrates that the IL-33/ST2 signaling pathway activates airway eosinophils that exacerbate airway inflammation in an autocrine and paracrine manner.

All solid organs contain resident monocyte-derived cells that appear early in organogenesis and persist throughout life. These cells are critical for normal development in some organs. Here we report the use of a previously described transgenic line, with EGFP driven by the macrophage-restricted Csf1r (c-fms) promoter, to image macrophage production and infiltration accompanying organogenesis in many tissues. Using microarray analysis of FACS-isolated EGFP-positive cells, we show that fetal kidney, lung and brain macrophages show similar gene expression profiles irrespective of their tissue of origin. EGFP-positive cells appeared in the renal interstitium from 12 days post coitum, prior to nephrogenesis, and maintain a close apposition to renal tubules postnatally. CSF-1 added to embryonic kidney explants increased overall renal growth and ureteric bud branching. Expression profiling of tissue macrophages and of CSF-1-treated explants showed evidence of the alternate, pro-proliferative (M2) activation profile, including expression of macrophage mannose receptor (CD206), macrophage scavenger receptor 2 (Msr2), C1q, CD163, selenoprotein P, CCL24 and TREM2. This response has been associated with the trophic role of tumour-associated macrophages. These findings suggest a trophic role of macrophages in embryonic kidney development, which may continue to play a similar role in postnatal repair.

Eosinophils have been implicated as playing a major role in allergic airway responses. However, the importance of these cells to the development of this disease has remained ambiguous despite many studies, partly because of lack of appropriate model systems. In this study, using transgenic murine models, we more clearly delineate a role for eosinophils in asthma. We report that, in contrast to results obtained on a BALB/c background, eosinophil-deficient C57BL/6 Delta dblGATA mice (eosinophil-null mice via the Delta DblGATA1 mutation) have reduced airway hyperresponsiveness, and cytokine production of interleukin (IL)-4, -5, and -13 in ovalbumin-induced allergic airway inflammation. This was caused by reduced T cell recruitment into the lung, as these mouse lungs had reduced expression of CCL7/MCP-3, CC11/eotaxin-1, and CCL24/eotaxin-2. Transferring eosinophils into these eosinophil-deficient mice and, more importantly, delivery of CCL11/eotaxin-1 into the lung during the development of this disease rescued lung T cell infiltration and airway inflammation when delivered together with allergen. These studies indicate that on the C57BL/6 background, eosinophils are integral to the development of airway allergic responses by modulating chemokine and/or cytokine production in the lung, leading to T cell recruitment.

Eotaxin and eotaxin-2, acting through CCR3, are potent eosinophil chemoattractants both in vitro and in animal models. In this study we examined the capacity of eotaxin and eotaxin-2 to recruit eosinophils and other inflammatory cells in vivo in human atopic and nonatopic skin. Skin biopsies taken after intradermal injection of eotaxin and eotaxin-2 were examined by immunohistochemistry. Allergen- and diluent-challenged sites were used as positive and negative controls. Eotaxin and eotaxin-2 produced a dose- and time-dependent local eosinophilia of comparable intensity in both atopic and nonatopic individuals. This was associated with an acute wheal and flare response at the site of injection and development of a cutaneous late phase reaction in a proportion of subjects. There was an accompanying decrease in mast cell numbers. Both chemokines also induced the accumulation of basophils and an unexpected early infiltration of neutrophils. Macrophages were prominent at the 24-h point. Although there was surface CCR3 expression on neutrophils in whole blood, we were unable to demonstrate any functional neutrophil responses to eotaxin in vitro. Thus, intradermal injection of eotaxin and eotaxin-2 in humans induced infiltration of eosinophils and other inflammatory cells as well as changes consistent with CC chemokine-induced mast cell degranulation.

OBJECTIVE

The importance of chemokines and their receptors to development and maintenance of allergic asthma is reflected in the burgeoning amount of literature currently devoted to this topic. Based on a series of selected references published during the last year, this review now summarizes recent advances and discusses the likely implications of these findings.

RESULTS

Of particular interest are reports describing novel interactions between chemokines and both eosinophils and mast cells, including a role for CXCL5 (epithelial cell-derived neutrophil-activating peptide-78) and intracellular CCR3. New insights into TH2-cell dominance are presented in reports dealing with a range of chemokines, including CCL3 (MIP-1alpha), CCL4 (MIP-1beta), CCL5 (RANTES), CXCL9 (Mig), and CXCL10 (IP-10). The increasing importance of structural cell participation is emphasized by reports focusing on the eotaxin family (CCL11, CCL24, and CCL26), as well as CCL17 (TARC), CCL22 (MDC), CXCL9 (Mig), and CX3CL1 (Fractalkine). A developing role for nonreceptor regulatory mechanisms is also emphasized by seminal work relating to metalloproteinases, as well as reports focusing on proteoglycans and beta-Arrestin-2. Finally, significant progress in the field of asthma heritability is featured in reports relating to both known and novel genes, including those encoding CCR5 and DPP-10.

CONCLUSIONS

The critical influence of chemokine biology on the outcome of allergic asthma continues to be highlighted in recent reports describing novel mechanisms by which eosinophils are recruited into the lung and local TH2-cell dominance is maintained. Also of considerable interest is the increasing emphasis currently being realized for structural cell participation, nonreceptor regulatory mechanisms, and the influence of susceptibility genes.

Clinical studies have demonstrated a link between the eosinophil-selective chemokines, eotaxins (eotaxin-1/CCL11 and eotaxin-2/CCL24), eosinophils, and the inflammatory bowel diseases, Crohn's disease and ulcerative colitis (UC). However, the cellular source and individual contribution of the eotaxins to colonic eosinophilic accumulation in inflammatory bowel diseases remain unclear. In this study we demonstrate, by gene array and quantitative PCR, elevated levels of eotaxin-1 mRNA in the rectosigmoid colon of pediatric UC patients. We show that elevated levels of eotaxin-1 mRNA positively correlated with rectosigmoid eosinophil numbers. Further, colonic eosinophils appeared to be degranulating, and the levels positively correlated with disease severity. Using the dextran sodium sulfate (DSS)-induced intestinal epithelial injury model, we show that DSS treatment of mice strongly induced colonic eotaxin-1 and eotaxin-2 expression and eosinophil levels. Analysis of eosinophil-deficient mice defined an effector role for eosinophils in disease pathology. DSS treatment of eotaxin-2(-/-) and eotaxin-1/2(-/-) mice demonstrated that eosinophil recruitment was dependent on eotaxin-1. In situ and immunofluorescence analysis-identified eotaxin-1 expression was restricted to intestinal F4/80(+)CD11b(+) macrophages in DSS-induced epithelial injury and to CD68(+) intestinal macrophages and the basolateral compartment of intestinal epithelial cells in pediatric UC. These data demonstrate that intestinal macrophage and epithelial cell-derived eotaxin-1 plays a critical role in the regulation of eosinophil recruitment in colonic eosinophilic disease such as pediatric UC and provides a basis for targeting the eosinophil/eotaxin-1 axis in UC.

The homing of proinflammatory (M1) and the "alternatively activated" anti-inflammatory (M2) macrophages plays a different role in the process of inflammation. Chemokines are the major mediators of macrophage chemotaxis, but how they differentially regulate M1 and M2 macrophages remains largely unclear. In the present study, we attempted to screen chemokines that differentially induce chemotaxis of M1 and M2 macrophages and to explore the underlying mechanism. Among the 41 chemokines that specifically bind to 20 chemokine receptors, CCL19, CCL21, CCL24, CCL25, CXCL8, CXCL10, and XCL2 specifically induced M1 macrophage chemotaxis, whereas CCL7 induced chemotaxis of both M1 and M2 macrophages. Whereas the differential effects of these chemokines on M1/M2 macrophage chemotaxis could be attributable to the predominant expression of their cognate receptors on the macrophage subsets, CCR7, the receptor for CCL19/CCL21, appeared to be an exception. Immunoblot analysis indicated an equivalent level of CCR7 in the whole cell lysate of M1 and M2 macrophages, but CCL19 and CCL21 only induced M1 macrophage chemotaxis. Both immunoblot and confocal microscopy analyses demonstrated that CCR7 was predominantly expressed on the cell surface of M1 but in the cytosol of M2 macrophages before ligand stimulation. As a result, CCL19 or CCL21 induced activation of both MEK1-ERK1/2 and PI3K-AKT cascades in M1 but not in M2 macrophages. Intriguingly, CCL19/CCL21-mediated M1 macrophage chemotaxis was blocked by specific inhibition of PI3K rather than MEK1. Together, these findings suggest that recruitment of M1 and M2 macrophages is fine tuned by different chemokines with the involvement of specific signaling pathways.

BACKGROUND

Allergic asthma is a chronic disease of the conducting airways characterized by T(H)2 inflammation and tissue remodeling after exposure to inhaled allergens. Although the T(H)2 profile is undisputed, the underlying molecular mechanisms leading to this abnormal T(H)2 profile remain largely unclear. MicroRNAs (miRNAs) are short noncoding RNAs that are important regulators of gene expression in the immune system. However, the role of miRNAs, specifically miR-155, in the regulation of allergic airway inflammation is unexplored.

OBJECTIVE

We sought to assess the contribution of miR-155 in a mouse model of allergic airway inflammation.

METHODS

To investigate a role for miR-155 in the regulation of allergic inflammation in vivo, we used miR-155 knockout (KO) and wild-type (WT) mice sensitized and exposed to ovalbumin.

RESULTS

miR-155 deficiency resulted in diminished eosinophilic inflammation and mucus hypersecretion in the lungs of allergen-sensitized and allergen-challenged mice compared with WT control animals. This was supported by a reduction in T(H)2 cell numbers and airway T(H)2 cytokine levels and complete abrogation of allergen-induced airway eotaxin-2/CCL24 and periostin levels in miR-155 KO mice. Intranasal instillation of eotaxin-2/CCL24 before allergen challenge partially restored airway eosinophilia in miR-155 KO mice, and adoptive transfer of CD4(+) T cells resulted in a similar degree of airway eosinophilia in miR-155 KO and WT mice. Furthermore, the transcription factor PU.1, a negative regulator of T(H)2 cytokine production, was upregulated in the airways of allergen-challenged miR-155 KO mice compared with WT mice.

CONCLUSIONS

Our data provides evidence that miR-155 contributes to the regulation of allergic airway inflammation by modulating T(H)2 responses through the transcription factor PU.1.

BACKGROUND

The cause of corticosteroid-resistant (CR) asthma is unknown.

OBJECTIVE

We sought to perform gene microarray analyses by using bronchoalveolar lavage (BAL) cells from well-characterized subjects with CR asthma and subject with corticosteroid-sensitive (CS) asthma to elucidate the differential expression of genes that contribute to the development of corticosteroid resistance.

METHODS

The patients were characterized as having CR or CS asthma based on FEV(1) percent predicted improvement after a 1-week course of oral prednisone. Expression of selected gene targets was verified by means of real-time PCR and ELISA.

RESULTS

Microarray analyses demonstrated significantly higher levels (>3-fold increase, P < .05) of transcripts for TNF-alpha, IL-1 alpha, IL-1 beta, IL-6, CXCL1, CXCL2, CXCL3, CXCL8 (IL-8), CCL3, CCL4, and CCL20 in BAL cells of subjects with CR asthma. These findings, confirmed by means of RT-PCR in additional BAL samples, were consistent with classical macrophage activation by bacterial products. In contrast, markers of alternatively activated macrophages, arginase I and CCL24, were decreased. Genes associated with activation of the LPS signaling pathway (early growth response 1, dual-specificity phosphatase 2, molecule possessing ankyrin repeats induced by LPS, and TNF-alpha-induced protein 3) were significantly increased in BAL samples from subjects with CR asthma (P < .05). These patients had significantly higher amounts (1444.0 +/- 457.3 pg/mg total protein) of LPS in BAL fluid than seen in subjects with CS asthma (270.5 +/- 216.0 pg, P < .05), as detected by using the LAL assay and confirmed by means of gas chromatographic/mass spectrometric analysis. Prolonged exposure to LPS induced functional steroid resistance to dexamethasone in normal human monocytes, as demonstrated by persistently increased IL-6 levels in the presence of dexamethasone.

CONCLUSIONS

Classical macrophage activation and induction of LPS signaling pathways along with high endotoxin levels detected in BAL fluid from subjects with CR asthma suggest that LPS exposure might contribute to CR asthma.

Pneumonia virus of mice (PVM; family Paramyxoviridae) is a natural pathogen of rodents that reproduces important clinical features of severe respiratory syncytial virus infection in humans. As anticipated, PVM infection induces transcription of IFN antiviral response genes preferentially in wild-type over IFN-alphabetaR gene-deleted (IFN-alphabetaR-/-) mice. However, we demonstrate that PVM infection results in enhanced expression of eotaxin-2 (CCL24), thymus and activation-regulated chemokine (CCL17), and the proinflammatory RNase mouse eosinophil-associated RNase (mEar) 11, and decreased expression of monocyte chemotactic protein-5, IFN-gamma-inducible protein-10, and TLR-3 in lung tissue of IFN-alphabetaR-/- mice when compared with wild type. No differential expression of chemokines MIP-1alpha or MIP-2 or Th2 cytokines IL-4 or IL-5 was observed. Differential expression of proinflammatory mediators was associated with distinct patterns of lung pathology. The widespread granulocytic infiltration and intra-alveolar edema observed in PVM-infected, wild-type mice are replaced with patchy, dense inflammatory foci localized to the periphery of the larger blood vessels. Bronchoalveolar lavage fluid from IFN-alphabetaR-/- mice yielded 7- to 8-fold fewer leukocytes overall, with increased percentages of eosinophils, monocytes, and CD4+ T cells, and decreased percentage of CD8+ T cells. Differential pathology is associated with prolonged survival of the IFN-alphabetaR-/- mice (50% survival at 10.8 +/- 0.6 days vs the wild type at 9.0 +/- 0.3 days; p < 0.02) despite increased virus titers. Overall, our findings serve to identify novel transcripts that are differentially expressed in the presence or absence of IFN-alphabetaR-mediated signaling, further elucidating interactions between the IFN and antiviral inflammatory responses in vivo.

Eotaxin-1/CCL11, eotaxin-2/CCL24, and eotaxin-3/CCL26 bind specifically and exclusively to CC chemokine receptor (CCR) 3, which is a potential therapeutic target in treating the peribronchial eosinophilia associated with allergic airway diseases. Bronchial epithelial cells represent an important source of chemokines, and thus we investigated in vitro and in vivo expression of eotaxin-2 and eotaxin-3 in bronchial epithelial cells in comparison with that of eotaxin-1. Immunohistochemistry showed increased expression of both eotaxin-2 and eotaxin-3 in addition to eotaxin-1 in asthmatics. Considerable amounts of eotaxins were secreted by bronchial epithelial lineage. As with eotaxin-1 production, generation of eotaxin-2 and eotaxin-3 by bronchial epithelial cells was up-regulated by IL-4 and IL-13, and attenuated by IFN-gamma and glucocorticoids. In addition to eotaxin-1 expression, but also eotaxin-2 and eotaxin-3 expression in the bronchial epithelium should be taken into consideration when developing the therapeutic strategies to treat eosinophilic airway diseases.

STAT6-mediated chemokine production in the lung is required for Th2 lymphocyte and eosinophil homing into the airways in allergic pulmonary inflammation, and thus is a potential therapeutic target in asthma. However, the critical cellular source of STAT6-mediated chemokine production has not been defined. In this study, we demonstrate that STAT6 in bone marrow-derived myeloid cells was sufficient for the production of CCL17, CCL22, CCL11, and CCL24 and for Th2 lymphocyte and eosinophil recruitment into the allergic airway. In contrast, STAT6 in airway-lining cells did not mediate chemokine production or support cellular recruitment. Selective depletion of CD11b(+) myeloid cells in the lung identified these cells as the critical cellular source for the chemokines CCL17 and CCL22. These data reveal that CD11b(+) myeloid cells in the lung help orchestrate the adaptive immune response in asthma, in part, through the production of STAT6-inducible chemokines and the recruitment of Th2 lymphocytes into the airway.

The chemokine receptor CXCR3 is predominantly expressed on T lymphocytes, and its agonists CXCL9, CXCL10 and CXCL11 are IFN-gamma-inducible chemokines that promote Th1 responses. In contrast, the CCR3 agonists CCL11, CCL24 and CCL26 are involved in the recruitment of cells such as eosinophils and basophils during Th2 responses. Here, we report that although CCL11, CCL24 and CCL26 are neither agonists nor antagonists of CXCR3, CCL11 binds with high affinity to CXCR3. This suggests that, in vivo, CXCR3 may act as a decoy receptor, sequestering locally produced CCL11. We also demonstrate that the CXCR3 ligands inhibit CCR3-mediated functional responses of both human eosinophils and CCR3 transfectants induced by all three eotaxins, with CXCL11 being the most efficacious antagonist. The examination of CCR3-CCR1 chimeric constructs revealed that CCL11 and CXCL11 share overlapping binding sites contained within the CCR3 extracellular loops, a region that was previously shown to be essential for effective receptor-activation. Hence, eosinophil responses mediated by chemokines acting at CCR3 may be regulated by two distinct mechanisms: the antagonistic effects of CXCR3 ligands and the sequestration of CCL11 by CXCR3-expressing cells. Such interplay may serve to finely tune inflammatory responses in vivo.

M2 macrophages have been subdivided into subtypes such as IL-4-induced M2a and IL-10-induced M2c in vitro. Although it was reported that IL-10 stimulation leads to an increase in IL-4Rα, the effect of IL-4 and IL-10 in combination with macrophage subtype differentiation remains unclear. Thus, we sought to clarify whether IL-10 enhanced the M2 phenotype induced by IL-4. In this study, we showed that IL-10 enhanced IL-4Rα expression in M-CSF-induced bone marrow-derived macrophages (BMDMs). Global gene expression analysis of M2 macrophages induced by IL-4, IL-10 or IL-4 + IL-10 showed that IL-10 enhanced gene expression of M2a markers induced by IL-4 in M-CSF-induced BMDMs. Moreover, IL-4 and IL-10 synergistically induced CCL24 (Eotaxin-2) production. Enhanced CCL24 expression was also observed in GM-CSF-induced BMDMs and zymosan-elicited, thioglycolate-elicited and naive peritoneal macrophages. CCL24 is a CCR3 agonist and an eosinophil chemoattractant. In vitro, IL-4 + IL-10-stimulated macrophages produced a large amount of CCL24 and increased eosinophil migration, which was inhibited by anti-CCL24 antibody. We also showed that IL-4 + IL-10-stimulated (but not IL-4 or IL-10 alone) macrophages transferred into the peritoneum of C57BL/6J mice increased eosinophil infiltration into the peritoneal cavity. These results demonstrate that IL-4 + IL-10-simulated macrophages have enhanced M2a macrophage-related gene expression, CCL24 production and eosinophil infiltration-inducing activity, thereby suggesting their contribution to eosinophil-related diseases.

Hypoxia is a condition of low oxygen tension occurring in inflammatory tissues. Dendritic cells (DC) are professional antigen-presenting cells whose differentiation, migration, and activities are intrinsically linked to the microenvironment. DCs will home and migrate through pathologic tissues before reaching their final destination in the lymph node. We studied the differentiation of human monocytes into immature DCs (iDCs) in a hypoxic microenvironment. We generated iDC in vitro under normoxic (iDCs) or hypoxic (Hi-DCs) conditions and examined the hypoxia-responsive element in the promoter, gene expression, and biochemical KEGG pathways. Hi-DCs had an interesting phenotype represented by up-regulation of genes associated with cell movement/migration. In addition, the Hi-DC cytokine/receptor pathway showed a dichotomy between down-regulated chemokines and up-regulated chemokine receptor mRNA expression. We showed that CCR3, CX3CR1, and CCR2 are hypoxia-inducible genes and that CCL18, CCL23, CCL26, CCL24, and CCL14 are inhibited by hypoxia. A strong chemotactic response to CCR2 and CXCR4 agonists distinguished Hi-DCs from iDCs at a functional level. The hypoxic microenvironment promotes the differentiation of Hi-DCs, which differs from iDCs for gene expression profile and function. The most prominent characteristic of Hi-DCs is the expression of a mobility/migratory rather than inflammatory phenotype. We speculate that Hi-DCs have the tendency to leave the hypoxic tissue and follow the chemokine gradient toward normoxic areas where they can mature and contribute to the inflammatory process.

Recently, eotaxin-CCR3 was reported to play an important role in choroidal neovascularization (CNV) development and was documented to be superior than vascular endothelial growth factor-A treatment when tested in CNV animals. As eotaxin studies are lacking in the human age-related macular degeneration (AMD) patients, we sought to determine whether eotaxin-2 (CCL24) has any association with inflammatory processes that occur in CNV. CCL24 levels were determined by enzyme linked immunosorbant assay (ELISA) after normalization to total serum protein and levels of ELISA were correlated to various risk factors in about 133 AMD patients and 80 healthy controls. The CCL24 levels were significantly higher in wet AMD patients as compared with dry AMD and normal controls. There was a significant difference when compared among wet AMD patients (i.e., minimally classic, predominantly classic, and occult). We also report significant difference in the CCL24 levels of Avastin-treated and untreated AMD patients. This study shows that CCL24 levels were found to be significantly increased in AMD patients despite Avastin treatment as compared with normal controls and those without Avastin, indicating that CCL24 may have an association with CNV and may be an important target to validate future therapeutic approaches in AMD in tandem with Avastin treatment.

New treatment approaches are needed for patients with asthma. Apolipoprotein A-I (apoA-I), the major structural protein of high-density lipoproteins, mediates reverse cholesterol transport and has atheroprotective and anti-inflammatory effects. In this study, we hypothesized that an apoA-I mimetic peptide might be effective at inhibiting asthmatic airway inflammation. A 5A peptide, which is a synthetic, bihelical apoA-I mimetic, was administered to wild-type A/J mice via osmotic mini-pump prior to the induction of house dust mite (HDM)-induced asthma. HDM-challenged mice that received the 5A apoA-I mimetic peptide had significant reductions in the number of bronchoalveolar lavage fluid eosinophils, lymphocytes, and neutrophils, as well as in histopathological evidence of airway inflammation. The reduction in airway inflammation was mediated by a reduction in the expression of Th2- and Th17-type cytokines, as well as in chemokines that promote T cell and eosinophil chemotaxis, including CCL7, CCL17, CCL11, and CCL24. Furthermore, the 5A apoA-I mimetic peptide inhibited the alternative activation of pulmonary macrophages in the lungs of HDM-challenged mice. It also abrogated the development of airway hyperresponsiveness and reduced several key features of airway remodeling, including goblet cell hyperplasia and the expression of collagen genes (Col1a1 and Col3a1). Our results demonstrate that the 5A apoA-I mimetic peptide attenuates the development of airway inflammation and airway hyperresponsiveness in an experimental murine model of HDM-induced asthma. These data support the conclusion that strategies using apoA-I mimetic peptides, such as 5A, might be developed further as a possible new treatment approach for asthma.

OBJECTIVE

The microfracture technique activates mesenchymal progenitors that enter the cartilage defect and form cartilage repair tissue. Synovial fluid (SF) has been shown to stimulate the migration of subchondral progenitors. The aim of our study was to determine the chemokine profile of SF from normal, rheumatoid arthritis (RA) and osteoarthritis (OA) donors and evaluate the chemotactic effect of selected chemokines on human subchondral progenitor cells.

METHODS

Chemokine levels of SF were analyzed using human chemokine antibody membrane arrays. The chemotactic potential of selected chemokines on human mesenchymal progenitors derived from subchondral cortico-spongious bone was tested using 96-well chemotaxis assays. Chemokine receptor expression of subchondral progenitors was assessed by real-time gene expression analysis and immuno-histochemistry.

RESULTS

Chemokine antibody array analysis showed that SF contains a broad range of chemokines. Ten chemokines that showed significantly reduced levels in RA or OA compared to normal SF or robustly high levels in all SF tested were used for further chemotactic analysis. Chemotaxis assays showed that the chemokines MDC/CCL22, CTACK/CCL27, ENA78/CXCL5 and SDF1α/CXCL12 significantly inhibited migration of progenitors, while TECK/CCL25, IP10/CXCL10 and Lymphotactin/XCL1 effectively stimulated cell migration. MCP1/CCL2, Eotaxin2/CCL24 and NAP2/CXCL7 showed no chemotactic effect on subchondral progenitors. Gene expression and immuno-histochemical analysis of corresponding chemokine receptors document presence of low levels of chemokine receptors in subchondral progenitors, with the CXCL10 receptor CXCR3 showing the highest expression level.

CONCLUSIONS

These results suggest that SF contains chemokines that may contribute to the recruitment of human mesenchymal progenitors from the subchondral bone in microfracture.

The pathophysiology of bipolar disorder (BD) includes, among other processes, changes in the neuroplasticity and regulation of apoptosis, which could potentially be influenced by inflammatory mediators such as chemokines. The objectives of this study were to investigate serum chemokine levels in patients with BD and to compare results with those obtained with healthy subjects. Here, serum chemokine levels of 30 euthymic patients with BD type I and 30 healthy volunteers were investigated and compared. The chemokines assessed were CCL2, CCL3, CCL8, CCL 9, CCL10, CCL11, and CCL24. Patients with BD showed significant differences in chemokine levels when compared with healthy subjects. While serum levels of CXCL10 were increased (p=.018), CCL24 levels were lower in bipolar patients (p=.025) when compared with controls. There was no statistical difference in the serum levels of CCL2, CCL3, CCL24, CXCL9, and CXCL11 between patients and controls. The presence of chemokine abnormalities in patients with BD during euthymia suggests that these inflammatory mediators should be further investigated with regard to their potential role as longstanding markers of the disorder.

The CD16(+) monocyte (Mo) subset produces proinflammatory cytokines and is expanded in peripheral blood during progression to AIDS, but its contribution to HIV pathogenesis is unclear. In this study, we investigate the capacity of human CD16(+) and CD16(-) Mo subsets to render resting CD4(+) T cells permissive for HIV replication. We demonstrate that CD16(+) Mo preferentially differentiate into macrophages (Mphi) that activate resting T cells for productive HIV infection by producing the CCR3 and CCR4 ligands CCL24, CCL2, CCL22, and CCL17. CD16(+), but not CD16(-), Mo-derived Mphi from HIV-infected and -uninfected individuals constitutively produce CCL24 and CCL2. Furthermore, these chemokines stimulate HIV replication in CD16(-) Mo:T cell cocultures. Engagement of CCR3 and CCR4 by CCL24 and CCL2, respectively, along with stimulation via CD3/CD28, renders T cells highly permissive for productive HIV infection. Moreover, HIV replicates preferentially in CCR3(+) and CCR4(+) T cells. These findings reveal a new pathway of T cell costimulation for increased susceptibility to HIV infection via engagement of CCR3 and CCR4 by chemokines constitutively produced by CD16(+) Mo/Mphi. Thus, expansion of CD16(+) Mo in peripheral blood of HIV-infected patients and their subsequent recruitment into tissues may contribute to chronic immune activation and establishment of viral reservoirs in resting T cells.

Eotaxin-3 (CCL26) belongs to the group of CC chemokines that attract eosinophils, basophils, and Th2 lymphocytes. Like eotaxin (CCL11) and eotaxin-2 (CCL24), eotaxin-3 mediates its activity through CCR3. Here we show that eotaxin-3 also binds to CCR2 on monocytes and CCR2-transfected cells. In contrast to monocyte chemotactic protein 1 (MCP-1; CCL2), eotaxin-3 does not trigger intracellular calcium mobilization, enzyme release, or phosphorylation of the mitogen-activated protein (MAP) kinase ERK and induces a weak chemotaxis in monocytes. Instead, eotaxin-3 inhibits MCP-1-mediated responses, thus acting as a natural antagonist for CCR2. This study also demonstrates that eotaxin-3 promotes active movement of monocytes away from a gradient of eotaxin-3 in vitro. This repellent effect is amplified when an additional gradient of MCP-1 is applied, demonstrating that the 2 mechanisms are synergistic. Eotaxin-3 effects on monocytes are largely abolished when cells are pretreated with MCP-1 or CCR2 antagonists. Like MCP-1-mediated migration, repulsion is sensitive to Bordetella pertussis toxin, indicating the involvement of Gi protein-coupled receptors. However, using transfected cells expressing CCR2 we could not detect F-actin formation or an active movement away induced by eotaxin-3, suggesting that either expression of a single receptor type is not sufficient to mediate cell repulsion or that the used transfected cell lines lack additional interaction molecules that are required for reverse migration. Eotaxin-3 was expressed by vascular endothelial cells and was essential for endothelial transmigration of eosinophils. Our data provide a mechanism by which 2 chemokine gradients that are oriented in opposite directions could cooperate in efficiently driving out monocytes from blood vessels into tissue.

Chronic airway disorders, including chronic obstructive pulmonary disease (COPD), cystic fibrosis, and asthma, are associated with persistent pulmonary inflammation and goblet cell metaplasia and contribute to significant morbidity and mortality worldwide. While the molecular pathogenesis of these disorders is actively studied, little is known regarding the transcriptional control of goblet cell differentiation and mucus hyperproduction. Herein, we demonstrated that pulmonary allergen sensitization induces expression of FOXM1 transcription factor in airway epithelial and inflammatory cells. Conditional deletion of the Foxm1 gene from either airway epithelium or myeloid inflammatory cells decreased goblet cell metaplasia, reduced lung inflammation, and decreased airway resistance in response to house dust mite allergen (HDM). FOXM1 induced goblet cell metaplasia and Muc5AC expression through the transcriptional activation of Spdef. FOXM1 deletion reduced expression of CCL11, CCL24, and the chemokine receptors CCR2 and CX3CR1, resulting in decreased recruitment of eosinophils and macrophages to the lung. Deletion of FOXM1 from dendritic cells impaired the uptake of HDM antigens and decreased cell surface expression of major histocompatibility complex II (MHC II) and costimulatory molecule CD86, decreasing production of Th2 cytokines by activated T cells. Finally, pharmacological inhibition of FOXM1 by ARF peptide prevented HDM-mediated pulmonary responses. FOXM1 regulates genes critical for allergen-induced lung inflammation and goblet cell metaplasia.

The proteomic analysis of bronchoalveolar lavage fluid (BALF) can give insight into pulmonary disease pathology and response to therapy. Here, we describe the first gel-free quantitative analysis of BALF in idiopathic pulmonary fibrosis (IPF), a chronic and fatal scarring lung disease. We utilized two-dimensional reversed-phase liquid chromatography and ion-mobility-assisted data-independent acquisition (HDMSE) for quantitation of >1000 proteins in immunodepleted BALF from the right middle and lower lobes of normal controls and patients with IPF. Among the analytes that were increased in IPF were well-described mediators of pulmonary fibrosis (osteopontin, MMP7, CXCL7, CCL18), eosinophil- and neutrophil-derived proteins, and proteins associated with fibroblast foci. For additional discovery and targeted validation, BALF was also screened by multiple reaction monitoring (MRM), using the JPT Cytokine SpikeMix library of >400 stable isotope-labeled peptides. A refined MRM assay confirmed the robust expression of osteopontin, and demonstrated, for the first time, upregulation of the pro-fibrotic cytokine, CCL24, in BALF in IPF. These results show the utility of BALF proteomics for the molecular profiling of fibrotic lung diseases and the targeted quantitation of soluble markers of IPF. More generally, this study addresses critical quality control measures that should be widely applicable to BALF profiling in pulmonary disease.