Mark C. Liu

Mepolizumab Treatment in Patients with Severe Eosinophilic Asthma

BACKGROUND Some patients with severe asthma have frequent exacerbations associated with persistent eosinophilic inflammation despite continuous treatment with high-dose inhaled glucocorticoids with or without oral glucocorticoids. METHODS In this randomized, double-blind, double-dummy study, we assigned 576 patients with recurrent asthma exacerbations and evidence of eosinophilic inflammation despite high doses of inhaled glucocorticoids to one of three study groups. Patients were assigned to receive mepolizumab, a humanized monoclonal antibody against interleukin-5, which was administered as either a 75-mg intravenous dose or a 100-mg subcutaneous dose, or placebo every 4 weeks for 32 weeks. The primary outcome was the rate of exacerbations. Other outcomes included the forced expiratory volume in 1 second (FEV1) and scores on the St. Georges Respiratory Questionnaire (SGRQ) and the 5-item Asthma Control Questionnaire (ACQ-5). Safety was also assessed. RESULTS The rate of exacerbations was reduced by 47% (95% confidence interval [CI], 29 to 61) among patients receiving intravenous mepolizumab and by 53% (95% CI, 37 to 65) among those receiving subcutaneous mepolizumab, as compared with those receiving placebo (P<0.001 for both comparisons). Exacerbations necessitating an emergency department visit or hospitalization were reduced by 32% in the group receiving intravenous mepolizumab and by 61% in the group receiving subcutaneous mepolizumab. At week 32, the mean increase from baseline in FEV1 was 100 ml greater in patients receiving intravenous mepolizumab than in those receiving placebo (P=0.02) and 98 ml greater in patients receiving subcutaneous mepolizumab than in those receiving placebo (P=0.03). The improvement from baseline in the SGRQ score was 6.4 points and 7.0 points greater in the intravenous and subcutaneous mepolizumab groups, respectively, than in the placebo group (minimal clinically important change, 4 points), and the improvement in the ACQ-5 score was 0.42 points and 0.44 points greater in the two mepolizumab groups, respectively, than in the placebo group (minimal clinically important change, 0.5 points) (P<0.001 for all comparisons). The safety profile of mepolizumab was similar to that of placebo. CONCLUSIONS Mepolizumab administered either intravenously or subcutaneously significantly reduced asthma exacerbations and was associated with improvements in markers of asthma control. (Funded by GlaxoSmithKline; MENSA ClinicalTrials.gov number, NCT01691521.).

Production of the novel C-C chemokine MCP-4 by airway cells and comparison of its biological activity to other C-C chemokines.

Monocyte chemotactic protein-4 (MCP-4) is a newly identified C-C chemokine with potent eosinophil chemoattractant properties. We describe studies of its biological activity in vitro to induce chemotaxis of peripheral blood eosinophils and to induce histamine release from IL-3-primed peripheral blood basophils. MCP-4 and eotaxin caused a similar rise in eosinophil intracytoplasmic Ca2+ and complete cross-desensitization. MCP-4 also abolished the eosinophil Ca2+ response to MCP-3 and partially desensitized the response to macrophage inflammatory protein-1alpha. MCP-4 activated cell migration via either CCR2b or CCR3 in mouse lymphoma cells transfected with these chemokine receptors. MCP-4 inhibited binding of 125I-eotaxin to eosinophils and CCR3-transfected cells and inhibited 125I-MCP-1 binding to CCR2b-transfectants. MCP-4 mRNA was found in cells collected in bronchoalveolar lavage of asthmatic and nonasthmatic subjects and was prominently expressed in human lung and heart. MCP-4 mRNA was expressed in several human bronchial epithelial cell lines after cytokine stimulation. Pretreatment of BEAS-2B epithelial cells with the glucocorticoid budesonide inhibited MCP-4 mRNA expression. These features make MCP-4 a candidate for playing a role in eosinophil recruitment during allergic respiratory diseases.

Identification of a novel cytokine, ML-1, and its expression in subjects with asthma.

A novel gene, designated ML-1, was identified from a human genomic DNA clone and human T cell cDNA sequences. The second exon of ML-1 gene shares significant sequence identity with the gene encoding IL-17 (IL-17). ML-1 gene expression was up-regulated in activated PBMCs, CD4+ T cells, allergen-specific Th0, Th1, and Th2 clones, activated basophils, and mast cells. Increased expression of the ML-1 gene, but not IL-17, was seen following allergen challenge in four asthmatic subjects, suggesting its role in allergic inflammatory responses. ML-1 from transiently transfected COS-7 cells was able to induce gene expression and protein production for IL-6 and IL-8 (at 10 ng/ml of ML-1: for IL-6, 599.6 ± 19.1 pg/ml; for IL-8, 1724.2 ± 132.9 pg/ml; and at 100 ng/ml of ML-1: for IL-6, 1005.3 ± 55.6 pg/ml; for IL-8, 4371.4 ± 280.5 pg/ml; p < 0.05 for both doses vs baseline) in primary bronchial epithelial (PBE) cells. Furthermore, increased expression of ICAM-1 was found in ML-1-stimulated PBE cells (mean fluorescence intensity (MFI) = 31.42 ± 4.39 vs baseline, MFI = 12.26 ± 1.77, p < 0.05), a functional feature distinct from IL-17 (MFI = 11.07 ± 1.22). This effect was not inhibited by a saturating amount of IL-17. These findings demonstrate that ML-1 is a novel cytokine with a distinct function, and suggest a different receptor for ML-1 on PBE cells.

A Genome-Wide Association Study on African-Ancestry Populations For Asthma

BACKGROUND Asthma is a complex disease characterized by striking ethnic disparities not explained entirely by environmental, social, cultural, or economic factors. Of the limited genetic studies performed on populations of African descent, notable differences in susceptibility allele frequencies have been observed. OBJECTIVES We sought to test the hypothesis that some genes might contribute to the profound disparities in asthma. METHODS We performed a genome-wide association study in 2 independent populations of African ancestry (935 African American asthmatic cases and control subjects from the Baltimore-Washington, DC, area and 929 African Caribbean asthmatic subjects and their family members from Barbados) to identify single-nucleotide polymorphisms (SNPs) associated with asthma. RESULTS A meta-analysis combining these 2 African-ancestry populations yielded 3 SNPs with a combined P value of less than 10(-5) in genes of potential biologic relevance to asthma and allergic disease: rs10515807, mapping to the alpha-1B-adrenergic receptor (ADRA1B) gene on chromosome 5q33 (3.57 x 10(-6)); rs6052761, mapping to the prion-related protein (PRNP) gene on chromosome 20pter-p12 (2.27 x 10(-6)); and rs1435879, mapping to the dipeptidyl peptidase 10 (DPP10) gene on chromosome 2q12.3-q14.2. The generalizability of these findings was tested in family and case-control panels of United Kingdom and German origin, respectively, but none of the associations observed in the African groups were replicated in these European studies. Evidence for association was also examined in 4 additional case-control studies of African Americans; however, none of the SNPs implicated in the discovery population were replicated. CONCLUSIONS This study illustrates the complexity of identifying true associations for a complex and heterogeneous disease, such as asthma, in admixed populations, especially populations of African descent.

TLR9- and FcεRI-Mediated Responses Oppose One Another in Plasmacytoid Dendritic Cells by Down-Regulating Receptor Expression

Plasmacytoid dendritic cells (pDC) express not only TLR9 molecules through which ligation with CpG DNA favors Th1 responses but also possess IgE receptors (FcεRI) implicated in allergen presentation and induction of Th2 responses. This dichotomy prompted an investigation to determine whether TLR9- and IgE receptor-mediated responses oppose one another in pDC by affecting receptor expression and associated functional responses. Results showed that IgE cross-linking reduced TLR9 in pDC and inhibited the capacity of these cells to secrete IFN-α when stimulated with the CpG oligodeoxynucleotide (ODN)-2216. In contrast, an ∼15-fold reduction in FcεRIα mRNA and a loss in surface protein were seen in pDC first exposed to TLR9 ligation with ODN-2216. Results indicated that type I IFNs partly mediated this effect, as rIFN-α also caused a significant ∼4-fold reduction in FcεRIα mRNA. Finally, this reduction in FcεRIα mediated by ODN-2216 correlated with a selective suppression of allergen-induced CD4+ T cell proliferation, but not of responses resulting from tetanus toxoid. Overall, these results imply mechanisms by which specific innate and IgE-dependent immune responses counterregulate one another at the dendritic cell level and may have significant impact on whether an ensuing response is either of Th1 or Th2 in nature.

Blood and bronchoalveolar eosinophils in allergic subjects after segmental antigen challenge: surface phenotype, density heterogeneity, and prostanoid production.

Eosinophil infiltration into the airways has been implicated in the pathophysiology of asthma. To improve our understanding of the function of eosinophils in asthma, we have compared the phenotype and function of eosinophils obtained simultaneously from blood and bronchoalveolar lavage (BAL) of allergic subjects 19 hours after segmental lung allergen challenge. Eosinophils were purified by discontinuous density gradient centrifugation, and their distribution at various layers was quantitated. Eosinophils at the 1.080 to 1.085 gm/ml interfaces from blood and BAL (purity > 70%) were analyzed by immunofluorescence and flow cytometry for several surface markers including adhesion-activation antigens. Eosinophils in BAL from antigen-challenged sites were markedly increased compared with control diluent-challenged BAL sites (0.3% +/- 1% vs 28.1% +/- 9.7%, n = 12, p < 0.002), and a greater percentage were hypodense (specific gravity < 1.080 gm/ml) than in peripheral blood (51.3 +/- 5.3 vs 19.0 +/- 4.4, n = 15, p < 0.01). In vitro, resting and activated BAL eosinophils biosynthesized less thromboxane B2 than blood eosinophils. Although both BAL and blood eosinophils expressed similar levels of Fc gamma RII (CD32), CD11a, and CD45, resting levels of Mo-1 (CD11b) were upregulated on BAL eosinophils (mean fluorescence intensity, 316% +/- 48% of blood eosinophils, n = 5, p < 0.05). Blood eosinophils stimulated in vitro with 1 mumol/L platelet activating factor or N-formyl-methionyl-leucyl-phenylalanine achieved levels of CD11b expression similar to those of BAL eosinophils. In contrast, CD11b expression on BAL eosinophils could not be further increased.(ABSTRACT TRUNCATED AT 250 WORDS)

Cutting edge: Expression of the C-C chemokine receptor CCR3 in human airway epithelial cells

Chemokine-induced eosinophil chemotaxis is mediated primarily through the C-C chemokine receptor, CCR3. We have now detected CCR3 immunoreactivity on epithelial cells in biopsies of patients with asthma and other respiratory diseases. CCR3 mRNA was detected by Northern blot analysis after TNF-α stimulation of the human primary bronchial epithelial cells as well as the epithelial cell line, BEAS-2B; IFN-γ potentiated the TNF-α-induced expression. Western blots and flow cytometry confirmed the expression of CCR3 protein. This receptor is functional based on studies demonstrating eotaxin-induced intracellular Ca2+ flux and tyrosine phosphorylation of cellular proteins. The specificity of this functional response was confirmed by blocking these signaling events with anti-CCR3 mAb (7B11) or pertussis toxin. Furthermore, 125I-eotaxin binding assay confirmed that CCR3 expressed on epithelial cells have the expected ligand specificity. These studies indicate that airway epithelial cells express CCR3 and suggest that CCR3 ligands may influence epithelial cell functions.

Serum Amyloid A Regulates Granulomatous Inflammation in Sarcoidosis through Toll-like Receptor-2

RATIONALE The critical innate immune mechanisms that regulate granulomatous inflammation in sarcoidosis are unknown. Because the granuloma-inducing component of sarcoidosis tissues has physicochemical properties similar to those of amyloid fibrils, we hypothesized that host proteins capable of forming poorly soluble aggregates or amyloid regulate inflammation in sarcoidosis. OBJECTIVES To determine the role of the amyloid precursor protein, serum amyloid A, as an innate regulator of granulomatous inflammation in sarcoidosis. METHODS Serum amyloid A expression was determined by immunohistochemistry in sarcoidosis and control tissues and by ELISA. The effect of serum amyloid A on nuclear factor (NF)-kappaB induction, cytokine expression, and Toll-like receptor-2 stimulation was determined with transformed human cell lines and bronchoalveolar lavage cells from patients with sarcoidosis. The effects of serum amyloid A on regulating helper T cell type 1 (Th1) granulomatous inflammation were determined in experimental models of sarcoidosis, using Mycobacterium tuberculosis catalase-peroxidase. MEASUREMENTS AND MAIN RESULTS We found that the intensity of expression and distribution of serum amyloid A within sarcoidosis granulomas was unlike that in many other granulomatous diseases. Serum amyloid A localized to macrophages and giant cells within sarcoidosis granulomas but correlated with CD3(+) lymphocytes, linking expression to local Th1 responses. Serum amyloid A activated NF-kappaB in Toll-like receptor-2-expressing human cell lines; regulated experimental Th1-mediated granulomatous inflammation through IFN-gamma, tumor necrosis factor, IL-10, and Toll-like receptor-2; and stimulated production of tumor necrosis factor, IL-10, and IL-18 in lung cells from patients with sarcoidosis, effects inhibited by blocking Toll-like receptor-2. CONCLUSIONS Serum amyloid A is a constituent and innate regulator of granulomatous inflammation in sarcoidosis through Toll-like receptor-2, providing a mechanism for chronic disease and new therapeutic targets.

Selective activation and accumulation of oligoclonal V beta-specific T cells in active pulmonary sarcoidosis.

Sarcoidosis is a granulomatous disease in which activated T cells, responding to an unidentified stimulus, accumulate at sites of disease such as the lung. To evaluate the hypothesis that active sarcoidosis is characterized by a selective activation and expansion of a limited repertoire of T cell receptor (TCR) specific T cells, we analyzed TCR V beta gene expression in lung and blood T cells of patients with active sarcoidosis and, for comparison, normal individuals using polymerase chain reaction amplification of 20 V beta gene families. Analysis of normal bronchoalveolar lavage T cells revealed TCR V beta distributions similar to that of normal blood, providing evidence for a lack of generalized skewing of the T cell repertoire in the normal, noninfected lung. Compared to normal lung and blood, subgroups of individuals with sarcoidosis demonstrated biased expression of one or more V beta genes in either the lung or blood. Five V beta gene families (V beta 5, V beta 8, V beta 15, V beta 16, and V beta 18) were most frequently utilized in a biased fashion by sarcoid lung or blood T cells. Furthermore, dramatic skewing of the T cell repertoire was apparent when sarcoid lung and blood T cells were expanded by short-term culture with IL-2. Sequence analysis demonstrated a bias in V beta gene expression was usually due to expansion of select V beta-specific clones, some of which contained a similar V(D)J junctional region motif. These observations provide evidence for a selective activation and accumulation of antigen-specific V beta-expressing T cells in sarcoidosis.

Effects of omalizumab on basophil and mast cell responses using an intranasal cat allergen challenge

BACKGROUND Omalizumab treatment suppresses FcepsilonRI expression faster on blood basophils than skin mast cells. OBJECTIVE We used omalizumab to elucidate the relative contributions of basophil versus mast cell FcepsilonRI activation in a nasal allergen challenge (NAC) model. METHODS Eighteen subjects with cat allergy were enrolled in a 3.5-month, double-blind, randomized (3.5:1), placebo-controlled trial of omalizumab using standard dosing. At baseline, subjects underwent NAC with lavage for prostaglandin D(2) measurement, skin prick test titration (SPTT), and blood sampling for basophil histamine release (BHR) and basophil IgE/FcepsilonRI measurements. Basophil studies were repeated at day 3 and then weekly until cat allergen-induced BHR was <20% of baseline or until day 45. Baseline visit procedures were repeated after the BHR reduction (midstudy NAC) and at the treatment periods completion (final NAC). RESULTS Subjects treated with omalizumab who completed all NACs (n = 12) demonstrated significant mean reduction in BHR to an optimal dose of cat allergen by midstudy NAC compared with baseline (74% decrease; P = .001). In addition, these subjects demonstrated significant decreases in mean combined nasal symptom scores (50% decrease; P = .007) and total sneeze counts (59% decrease; P = .01) by midstudy NAC relative to baseline NAC. In contrast, measures of mast cell response (SPTT and nasal lavage prostaglandin D(2)) were only significantly reduced by the final NAC. Subjects on placebo (n = 4) did not experience a shift in basophil, NAC symptom, or mast cell measures. CONCLUSION Reduction in nasal symptom scores occurred when the basophil, but not mast cell, response was reduced on omalizumab, implicating a role for basophils in the acute NAC response.