Sanjiv Sur

IFN-γ-Inducing Factor (IL-18) Increases Allergic Sensitization, Serum IgE, Th2 Cytokines, and Airway Eosinophilia in a Mouse Model of Allergic Asthma

We investigated the effects of IFN-γ-inducing factor (IL-18) in a ragweed (RW) mouse model of allergic asthma. Administration of IL-18 in conjunction with allergic sensitization and challenge in wild-type, but not IFN-γ −/− mice, inhibited the bronchoalveolar lavage (BAL) eosinophilia induced by RW challenge, and increased serum levels of RW-specific IgG2a and production of IFN-γ from splenocytes cultured with RW, indicating a critical role for IFN-γ in mediating these effects. Paradoxically, the same treatment schedule in WT mice increased serum levels of RW-specific IgE and IgG1, and production of IL-4 and IL-5 from splenocytes cultured with RW. When the effects of the same IL-18 treatment schedule were allowed to mature for 3 wk, the inhibition of lung eosinophil recruitment was replaced by augmentation of lung eosinophil recruitment. In another experiment, IL-18 administered only with allergic sensitization increased BAL eosinophilia and lung expression of IL-5 and IFN-γ, while IL-18 administered only with RW challenge decreased BAL eosinophilia and increased lung IFN-γ expression, while lung expression of IL-5 remained unchanged. IL-18 administered without RW or adjuvant to naive mice increased total serum IgE levels. Finally, intrapulmonary administrations of IL-18 plus RW in naive mice dramatically increased Th2 cytokine production, IgE levels, eosinophil recruitment, and airway mucus, demonstrating induction of allergic sensitization. This is the first report demonstrating that IL-18 promotes a Th2 phenotype in vivo, and potently induces allergic sensitization. These results suggest that IL-18 may contribute to the pathogenesis of allergic asthma.

Localization of eosinophil-derived neurotoxin and eosinophil cationic protein in neutrophilic leukocytes.

Eosinophil‐derived neurotoxin (EDN) and eosinophil cationic protein (ECP) are generally regarded as eosinophil‐specific proteins. We tested whether EDN and ECP are present in mature neutrophils. By indirect immunofluorescence, both eosinophils and neutrophils stained with antibodies to EDN and ECP. Lysates of purified (<0.1% eosinophil contamination) neutrophils contained EDN, 112 ± 4 ng/106 cells, and ECP, 163 ± 2 ng/106 cells, whereas eosinophil major basic protein (MBP) was not detectable. Electron microscopic examination of immunogold‐labeled buffy coat cells stained with EDN antibody showed that EDN is localized to neutrophil granules. Finally, EDN mRNA was detected in lysates of highly purified neutrophils (0.001% eosinophil contamination) by the reverse transcription‐polymerase chain reaction. We conclude that proteins that are either identical to or immunologically cross‐reactive with EDN and ECP are present in neutrophils and that EDN is synthesized and localized to neutrophil granules. Thus, caution must be exercised in interpreting the presence of EDN and ECP as specific markers of eosinophil‐associated inflammation in human disease. J. Leukoc. Biol. 63: 715–722; 1998.

Assessment of eosinophil and neutrophil participation in atopic dermatitis: Comparison with the IgE-mediated late-phase reaction

We hypothesized that repeated IgE-mediated late-phase reactions are critical in the pathogenesis of atopic dermatitis (AD). Prior studies have shown that extracellular deposition of eosinophil granule major basic protein (MBP) occurs in lesional AD skin, despite a paucity of infiltrating eosinophils, and that deposition of both neutrophil and eosinophil granule proteins occurs in the IgE-mediated late-phase reaction. We evaluated the participation of both eosinophil and neutrophil granule proteins in AD. Cutaneous biopsy specimens and serum and urine samples were obtained from 22 patients with AD. Lesional tissue was examined by means of immunofluorescence for neutrophil elastase and lactoferrin and for eosinophil granule MBP, eosinophil-derived neurotoxin (EDN), and eosinophil cationic protein (ECP). Serum levels of elastase, MBP, EDN, and ECP and urine levels of MBP, EDN, and ECP were measured. Marked extracellular deposition of at least one of the eosinophil granule proteins was present in the dermis of 15 of the 22 AD skin specimens, but minimal or no extracellular neutrophil elastase or lactoferrin deposition was observed in any specimens. Serum and urine levels of MBP, EDN, and ECP in the patients were elevated when compared with those of normal controls, whereas serum levels of neutrophil elastase were not elevated. Serum MPB levels correlated with extent of body surface involvement. These results suggest that eosinophil degranulation occurs in AD but that neutrophil degranulation does not. Although eosinophil degranulation is prominent in both the late-phase reaction and in AD, the lack of neutrophil degranulation in AD demonstrates differences in the inflammatory reactions.

CCL7 and CXCL10 Orchestrate Oxidative Stress-Induced Neutrophilic Lung Inflammation

Oxidative stress from ozone (O3) exposure augments airway neutrophil recruitment and chemokine production. We and others have shown that severe and sudden asthma is associated with airway neutrophilia, and that O3 oxidative stress is likely to augment neutrophilic airway inflammation in severe asthma. However, very little is known about chemokines that orchestrate oxidative stress-induced neutrophilic airway inflammation in vivo. To identify these chemokines, three groups of BALB/c mice were exposed to sham air, 0.2 ppm O3, or 0.8 ppm O3 for 6 h. Compared with sham air, 0.8 ppm O3, but not 0.2 ppm O3, induced pronounced neutrophilic airway inflammation that peaked at 18 h postexposure. The 0.8 ppm O3 up-regulated lung mRNA of CXCL1,2,3 (mouse growth-related oncogene-α and macrophage-inflammatory protein-2), CXCL10 (IFN-γ-inducible protein-10), CCL3 (macrophage-inflammatory protein-1α), CCL7 (monocyte chemoattractant protein-3), and CCL11 (eotaxin) at 0 h postexposure, and expression of CXCL10, CCL3, and CCL7 mRNA was sustained 18 h postexposure. O3 increased lung protein levels of CXCL10, CCL7, and CCR3 (CCL7R). The airway epithelium was identified as a source of CCL7. The role of up-regulated chemokines was determined by administering control IgG or IgG Abs against six murine chemokines before O3 exposure. As expected, anti-mouse growth-related oncogene-α inhibited neutrophil recruitment. Surprisingly, Abs to CCL7 and CXCL10 also decreased neutrophil recruitment by 63 and 72%, respectively. These findings indicate that CCL7 and CXCL10, two chemokines not previously reported to orchestrate neutrophilic inflammation, play a critical role in mediating oxidative stress-induced neutrophilic airway inflammation. These observations may have relevance in induction of neutrophilia in severe asthma.

The Differential Role of Extracellular Signal-Regulated Kinases and p38 Mitogen-Activated Protein Kinase in Eosinophil Functions

The activation of eosinophils by cytokines is a major event in the pathogenesis of allergic diseases. We have investigated the activation of mitogen-activated protein (MAP) kinases and their functional relevance in eosinophil differentiation, survival, degranulation, and cytokine production. IL-5 induced phosphorylation and activation of extracellular signal-regulated kinases (ERK) and p38 MAP kinases in eosinophils. PD98059, a MAP/ERK kinase inhibitor, blocked phosphorylation of ERK1/2 in a dose-dependent manner. SB202190, a p38 inhibitor, blocked p38-dependent phosphorylation of activating transcription factor-2. To study the importance of the MAP kinases on eosinophil differentiation, we cultured mouse bone marrow cells with IL-3 and IL-5 in the presence of the inhibitors. SB202190 dramatically inhibited eosinophil differentiation by 71%. PD98059 was less potent and reduced eosinophil differentiation by 28%. Both inhibitors marginally inhibited eosinophil survival only at the highest doses. Prolonged incubation of eosinophils with IL-5 induced significant eosinophil-derived neurotoxin release. Both PD98059 and SB202190 nearly completely inhibited (87% and 100% inhibition, respectively) IL-5-stimulated eosinophil-derived neurotoxin release in a dose-dependent manner. Next, we examined the effect of the MAP kinase inhibitors on eosinophil production of the cytokine macrophage-inflammatory protein (MIP)-1α. PD98059 blocked C5a- but not ionomycin-induced MIP-1α production (59% inhibition at 50 μM concentration). In contrast, SB202190 nearly completely inhibited (99%) C5a-induced MIP-1α production. Further, it blocked ionomycin-stimulated production by 66%. Our results suggest that both p38 and ERK1/2 MAP kinases play an important role in eosinophil differentiation, cytokine production, and degranulation. The p38 MAP kinase plays a greater role than ERK1/2 in eosinophil differentiation and cytokine production.

Unc119, a Novel Activator of Lck/Fyn, Is Essential for T Cell Activation

The first step in T cell receptor for antigen (TCR) signaling is the activation of the receptor-bound Src kinases, Lck and Fyn. The exact mechanism of this process is unknown. Here, we report that the novel Src homology (SH) 3/SH2 ligand–Uncoordinated 119 (Unc119) associates with CD3 and CD4, and activates Lck and Fyn. Unc119 overexpression increases Lck/Fyn activity in T cells. In Unc119-deficient T cells, Lck/Fyn activity is dramatically reduced with concomitant decrease in interleukin 2 production and cellular proliferation. Reconstitution of cells with Unc119 reverses the signaling and functional outcome. Thus, Unc119 is a receptor-associated activator of Src-type kinases. It provides a novel mechanism of signal generation in the TCR complex.

Eosinophilic inflammation is associated with elevation of interleukin-5 in the airways of patients with spontaneous symptomatic asthma ☆ ☆☆ ★ ★★

In vitro and in vivo studies have shown an important role for interleukin-5 (IL-5) in regulating eosinophil proliferation, survival, and effector function. Because eosinophilic inflammation is an important component of symptomatic episodes of asthma, we have investigated whether increased levels of IL-5 protein are present in bronchoalveolar lavage (BAL) fluid of patients with spontaneously symptomatic asthma (FEV1, l61% predicted; FEF25%-75%, 30% predicted) compared with patients with asymptomatic asthma (FEV1, 88% predicted; FEF25%-l75%, 76% predicted). The percent of BAL eosinophils (10.5% vs 0.6]) (p = 0.0001) and eosinophil-derived neurotoxin (386.0 ng/ml vs 6.3 ng/ml) (p = 0.0001) was greater in BAL fluids derived from patients with symptomatic asthma compared with patients with asymptomatic asthma. Levels of IL-5 measured with an immunoradiometric assay were significantly higher in patients with symptomatic asthma (n = 26) compared with those with asymptomatic asthma (n = 18) (274 pg/ml vs < pg/ml) (p = 0.02). The increased IL-5 levels were noted in a subset of patients with symptomatic asthma with BAL absolute eosinophil counts greater than 10(6) (IL-5, 664 pg/ml; n = 10) as opposed to patients with symptomatic asthma with BAL eosinophil counts less than 10(6) (IL-5, < 13 pg/ml; n = 16) (p = 0.005). This study suggests that IL-5 is not only induced in experimental models of allergen-induced asthma but can also be detected as asthma progresses from the asymptomatic to the clinically symptomatic state in subjects with significant BAL eosinophilia.

Eosinophil survival activity identified as interleukin-5 is associated with eosinophil recruitment and degranulation and lung injury twenty-four hours after segmental antigen lung challenge

BACKGROUND Segmental antigen challenge in allergic volunteer subjects leads to the recruitment of inflammatory cells, including eosinophils, to the lung and to lung injury as shown by albumin influx into the alveolar air space. The goal of this study was to determine whether eosinophil-active cytokines, including IL-3, IL-5, or granulocyte-macrophage colony-stimulating factor, are released into the lung 24 hours after segmental antigen challenge of ragweed allergic subjects with allergic rhinitis and to determine whether the presence of the cytokine or cytokines is correlated with markers of lung inflammation and lung injury. METHODS Volunteers underwent challenge with a wide variety of antigen doses, which resulted in the recruitment of inflammatory cell mixtures both with and without eosinophils. RESULTS Eosinophil survival activity (ESA), the ability of the cytokine to prolong blood eosinophil survival in culture, was found in 5 of 17 ragweed allergic subjects and only in subjects challenged with relatively high doses of ragweed antigen (0.2 ragweed antigen units/ml or more). No ESA was found in bronchoalveolar lavage (BAL) fluid in any of eight nonragweed allergic subjects. This activity could be almost completely neutralized by preincubating BAL fluid with specific antibody to IL-5, although a small contribution by granulocyte-macrophage colony-stimulating factor may also have been present. ESA correlated with eosinophil recruitment (r = 0.72, p < 0.001) and degranulation in the lung (r = 0.63 to 0.81, p < 0.01, for eosinophil granule constituents in BAL fluid) and lung injury as shown by albumin influx into the alveolar air spaces (r = 0.83, p < 0.001). ESA was unrelated to the presence of other inflammatory cells in the lung. Subjects who had IL-5 in BAL fluid appeared to undergo more severe initial reactions to antigen challenge. CONCLUSIONS We conclude that IL-5 is the most important constituent in ESA in the lung 24 hours after antigen challenge and that it correlates with eosinophil recruitment, degranulation, and lung injury.

Eosinophil recruitment is associated with IL-5, but not with RANTES, twenty-four hours after allergen challenge

Several lines of evidence suggest that the chemokine RANTES may play a role in eosinophilia observed during allergic inflammation. To test this hypothesis, six patients with allergic asthma were studied. After performing bronchoalveolar lavage in a lung segment (baseline), segmental bronchoprovocation was performed with saline solution in another segment and with ragweed in a third segment. Bronchoalveolar lavage was performed 24 hours later in the saline-challenged (sham) and ragweed-challenged lung segments. The bronchoalveolar lavage fluids from the baseline, sham, and ragweed segments were analyzed for cell counts and for the levels of IL-5, RANTES, and eosinophil-derived neurotoxin. IL-5 levels were elevated in the ragweed (984 +/- 588 pg/ml) compared with sham segments (2.8 +/- 0.2 pg/ml, p = 0.02). Likewise, RANTES levels were elevated in the ragweed (12.93 +/- 3.4 pg/ml) compared with the sham segments (3.05 +/- 1.19 pg/ml, p = 0.006). The IL-5 levels correlated with both eosinophil numbers (r = 0.90, p < 0.02) and eosinophil-derived neurotoxin levels (r = 0.89, p < 0.02). In contrast, RANTES levels did not correlate with either eosinophil numbers or eosinophil-derived neurotoxin levels. These results indicate that although both IL-5 and RANTES are elevated 24 hours after allergen challenge, only IL-5 correlates with eosinophil recruitment and degranulation.

Immunofluorescent staining for mast cells in idiopathic pulmonary fibrosis: quantification and evidence for extracellular release of mast cell tryptase

In many diseases, retrospective analysis for determining the presence of mast cells has been difficult because of their loss of metachromatic staining properties once tissue has undergone formalin fixation. We quantified mast cells in peribronchiolar tissue of idiopathic pulmonary fibrosis (IPF) and in normal human lung by using rabbit antiserum to human mast cell tryptase. In lung biopsy specimens from 15 patients with IPF, the mean number of mast cells per high-power field in connective tissue directly adjacent to the lumen of small airways (0.5 to 2 mm in diameter) and other fibrotic foci was 29.9 +/- 10.8 in comparison with 13.7 +/- 3.5 in 16 normal controls (P < 0.001). In addition, mast cells in cases of IPF had an altered appearance--irregularity of the plasma membrane and release of extracellular tryptase. We conclude that the number of mast cells is increased in IPF and that the altered appearance of the mast cells suggests that they are activated and undergoing degranulation.