James L. Checkel

Eosinophil granule proteins in peripheral blood granulocytes.

Eosinophils contain four principal cationic proteins, major basic protein (MBP), eosinophil‐derived neurotoxin (EDN), eosinophil cationic protein (ECP), and eosinophil peroxidase (EPO). To determine the quantities of these proteins in granulocytes and whether they are specific to eosinophils, their concentrations in ly‐ sates of human granulocytes were measured using specific radioimmunoassays. The effect of different methods for eosinophil lysis on the recovery of the proteins was also studied. Maximal recovery occurred at pH 2 for MBP and pH 5.6 for the other granule proteins. The proteins cosedimented with eosinophils and their concentrations (X ± SEM) in ng/106 eosinophils (and in nM/106 eosinophils) were: MBP, 8,982 ± 611 (641.6); EDN, 3,283 ± 116 (178.4); ECP, 5,269 ± 283 (250.9); and EPO, 12,174 ± 859 (171.5). Basophils from a normal person contained (in ng/106 cells) MBP, 2,374; EDN, 214; ECP, 77; and EPO, 17. Highly purified neutrophils contained (in ng/106 cells) MBP, 3 ± 0.5; EDN, 72 ± 9; and ECP, 50 ± 12. Therefore we conclude that these proteins are mainly expressed in eosinophils, but that certain ones are present in basophils and neutrophils.

Acidic polyamino acids inhibit human eosinophil granule major basic protein toxicity. Evidence of a functional role for ProMBP.

Eosinophil granule major basic protein (MBP), a potent toxin for helminths and mammalian cells in vitro, is a single polypeptide chain rich in arginine. MBP has been localized on damaged helminths and tissues in hypersensitivity diseases including bronchial asthma. The MBP cDNA indicates that MBP is translated as a slightly acidic preproprotein with an acidic propart. To test the hypothesis that the acidic pro-part of proMBP inhibits the toxicity of mature MBP, acidic polyamino acids (aa) were used as antagonists of MBP toxicity to K562 cells and guinea pig tracheal epithelium and used as antagonists of MBP airway hyperresponsiveness in primates. The acidic poly aa inhibited MBP toxicity and MBP airway hyperresposiveness. The acidic poly aa inhibited MBP toxicity in a charge-dependent manner similar to that proposed for proMBP, suggesting that the acidic pro-part of proMBP functions to mask mature MBP toxicity. This inhibition was not limited to MBP, but also applied to polyarginine and eosinophil cationic protein. These acidic poly aa may be useful to inhibit the actions of a number of cationic toxins released by the eosinophil in numerous hypersensitivity diseases.

RECOGNITION OF FUNGAL PROTEASE ACTIVITIES INDUCES CELLULAR ACTIVATION AND EOSINOPHIL-DERIVED NEUROTOXIN RELEASE IN HUMAN EOSINOPHILS

Eosinophils are multifunctional leukocytes implicated in the pathogenesis of asthma and in immunity to certain organisms. Associations between exposure to an environmental fungus, such as Alternaria, and asthma have been recognized clinically. Protease-activated receptors (PARs) are G protein-coupled receptors that are cleaved and activated by serine proteases, but their roles in innate immunity remain unknown. We previously found that human eosinophils respond vigorously to Alternaria organisms and to the secretory product(s) of Alternaria with eosinophils releasing their proinflammatory mediators. In this study, we investigated the roles of protease(s) produced by Alternaria and of PARs expressed on eosinophils in their immune responses against fungal organisms. We found that Alternaria alternata produces aspartate protease(s) and that human peripheral blood eosinophils degranulate in response to the cell-free extract of A. alternata. Eosinophils showed an increased intracellular calcium concentration in response to Alternaria that was desensitized by peptide and protease ligands for PAR-2 and inhibited by a PAR-2 antagonistic peptide. Alternaria-derived aspartate protease(s) cleaved PAR-2 to expose neo-ligands; these neo-ligands activated eosinophil degranulation in the absence of proteases. Finally, treatment of Alternaria extract with aspartate protease inhibitors, which are conventionally used for HIV-1 and other microbes, attenuated the eosinophils’ responses to Alternaria. Thus, fungal aspartate protease and eosinophil PAR-2 appear critical for the eosinophils’ innate immune response to certain fungi, suggesting a novel mechanism for pathologic inflammation in asthma and for host-pathogen interaction.

Post-translational Tyrosine Nitration of Eosinophil Granule Toxins Mediated by Eosinophil Peroxidase

Nitration of tyrosine residues has been observed during various acute and chronic inflammatory diseases. However, the mechanism of tyrosine nitration and the nature of the proteins that become tyrosine nitrated during inflammation remain unclear. Here we show that eosinophils but not other cell types including neutrophils contain nitrotyrosine-positive proteins in specific granules. Furthermore, we demonstrate that the human eosinophil toxins, eosinophil peroxidase (EPO), major basic protein, eosinophil-derived neurotoxin (EDN) and eosinophil cationic protein (ECP), and the respective murine toxins, are post-translationally modified by nitration at tyrosine residues during cell maturation. High resolution affinity-mass spectrometry identified specific single nitration sites at Tyr349 in EPO and Tyr33 in both ECP and EDN. ECP and EDN crystal structures revealed and EPO structure modeling suggested that the nitrated tyrosine residues in the toxins are surface exposed. Studies in EPO-/-, gp91phox-/-, and NOS-/- mice revealed that tyrosine nitration of these toxins is mediated by EPO in the presence of hydrogen peroxide and minute amounts of NOx. Tyrosine nitration of eosinophil granule toxins occurs during maturation of eosinophils, independent of inflammation. These results provide evidence that post-translational tyrosine nitration is unique to eosinophils.

IL-1 Family Cytokines Drive Th2 and Th17 Cells to Innocuous Airborne Antigens

Allergic asthma is commonly thought to result from dysregulated airway inflammatory responses to ubiquitous environmental antigens mediated by CD4(+) T cells polarized to a Th2 or Th17 cell. However, the mechanisms involved in the development of these T-cell responses remain unknown. This study examines the effects of IL-1 family cytokines, such as IL-33 and IL-1β, on the development of antigen-specific Th2 and Th17 cells in the airway. We administered IL-1 family cytokines and model antigens, such as ovalbumin, into the airways of naive BALB/c mice, and examined the cellular and humoral immune responses. To investigate the immunologic mechanisms, we used IL-4 green fluorescent protein reporter mice and mice deficient in the Il4 gene. Innocuous antigens, such as endotoxin-free ovalbumin and short ragweed extract, did not sensitize naive mice when administered through the airways. However, when mice were exposed to the same antigens with IL-1β or IL-33, they developed IgE antibodies. In particular, IL-33 induced robust and long-lasting Th2 cells that produced a large quantity of IL-5 and IL-13 and asthma-like airway pathology. IL-1β induced Th17 cells. In naive, nonsensitized animals, IL-33 stimulated endogenous IL-4 expression by CD4(+) T cells, which was critical for the polarization of CD4(+) T cells to the Th2 type. In the absence of IL-4, mice developed Th17 cells and neutrophilic airway inflammation. In conclusion, IL-1 family cytokines possess a potent adjuvant activity to promote both Th2 and Th17 cells to innocuous airborne antigens, and they may play fundamental roles in the immunopathology of asthma.

Inflammatory responses of human eosinophils to cockroach are mediated through protease-dependent pathways

To the Editor: Exposure to cockroach is one of the major risk factors for developing asthma (1). Among inner-city children, cockroach allergen has an important role in increasing asthma morbidity. The molecular mechanism for this association between cockroach exposure and asthma is unclear. Cockroaches are complex organisms, and the presence and activities of proteases in cockroach extracts have been controversial. In general, the individual allergen proteins, whether purified or recombinant, do not show proteolytic activity (2). However, protease activities were detected in German cockroach frass and whole body extract (3). Eosinophils are likely involved in the pathophysiology of asthma, atopic dermatitis, and certain gastrointestinal diseases (4). Herein, we examined the effects of cockroach products on the activation and effector functions of human eosinophils, and we investigated the biologically active molecules in cockroach extract. As previously described (5), 12.5 μg/ml German or 6.2 μg/ml Oriental cockroach extracts incubated for 3 h with isolated eosinophils released the eosinophil granule protein, eosinophil-derived neurotoxin (EDN), (see Figure E1 in this article’s Online Repository at www.jacionline.org). German cockroach extract induced superoxide anion production after 15 minutes of exposure, and Oriental cockroach extract induced superoxide anion production after 30 minutes (Figure E1). Both German and Oriental cockroach extracts treated at 4 °C or 37 °C potently induced EDN release (Figure 1A). In contrast, inactivating the extracts at 56 °C decreased their ability to stimulate eosinophils (p<0.05, n=4), and treatment at 100 °C almost abolished the activity. To verify the importance of heat-labile molecule(s) in eosinophil activation induced by cockroach extracts and to demonstrate their lack of direct toxicity to eosinophils, we examined IL-8 production. After 24 hr, both German and Oriental cockroach extracts induced IL-8 production by eosinophils (p<0.05, n=4) (Figure 1B); their stimulatory effects were partially heat-inactivated at 56 °C and abolished at 100 °C. Thus, heat-labile molecules, likely proteases, in cockroach extracts are involved in eosinophil activation. Figure 1 The eosinophil-stimulating activities of cockroach extracts are heat-sensitive PARs, in particular PAR-2, likely play a major role in eosinophil activation in response to proteases (6). Therefore, we investigated whether cockroach extracts contain proteolytic activities that activate PAR-2. An authentic ligand for PAR-2, namely trypsin, cleaves the extracellular N-terminus of PAR-2 between R36 and S37 and exposes a tethered “neo-ligand” (i.e. S37LIGKV-) that binds intramolecularly to PAR-2 and triggers cellular activation (7). To mimic the proteolytic activation of PAR-2, we used a fluorogenic peptide substrate, Abz-SKGRSLIGKdD, which encompasses the trypsin cleavage site of human PAR-2 (from Ser33 to Lys41) (8); the Abz group fluoresces only after release of the KdD group, following cleavage of internal peptides. As expected, trypsin cleaved the PAR-2 peptide, increasing the fluorescence intensity over time (Figure 2A). German and Oriental cockroach extracts also cleaved the PAR-2 peptide in a concentration-dependent manner. To characterize the cockroach proteases cleaving this PAR-2, we used protease inhibitors. Pepstatin A specifically inhibits acid proteases, in particular aspartate proteases. Absorption of a complex protease mixture with pepstatin A agarose removes aspartate-like proteases. Treatment of German and Oriental cockroach extracts with pepstatin A agarose decreased PAR-2 cleavage by 70% and 55 %, respectively (p<0.05, n=5); treatment with control agarose showed no effects (Figure 2B). Pepstatin A treatment showed no effects on trypsin-mediated PAR-2 cleavage, demonstrating the inhibitor’s specificity. A serine protease inhibitor, APMSF, and a cysteine protease inhibitor, E64, did not inhibit PAR-2 cleavage by cockroach extracts (Figure 2C); APMSF significantly inhibited trypsin-induced PAR-2 cleavage. Furthermore, after pretreatment with pepstatin A, both German and Oriental cockroach extracts induced less EDN release compared to the untreated extracts (Figure 2D, p<0.05, n=5). Pretreatment of PMA with pepstatin A did not affect eosinophil degranulation. In addition, preincubating eosinophils with the PAR-2 antagonistic peptide, LSIGKV, before exposure to German cockroach extract inhibited degranulation by an average of 37% (p<0.01, n=9). Overall, the cockroach extracts likely contain pepstatin A-sensitive protease(s) that activate PAR-2 and induce eosinophil degranulation. Figure 2 Cockroach extracts have pepstatin-sensitive aspartate protease activity that is involved in PAR-2 cleavage and eosinophil degranulation In general, trypsin and trypsin-like proteases cleave PAR-2 specifically between Arg36 and Ser37 (7). To investigate the PAR-2 site cleaved by cockroach extracts, we analyzed the peptide fragments by reverse-phase-HPLC-ESI-MS. The same peptide substrate, as used in the PAR-2 cleavage enzymatic assay (Abz-SKGR36S37LIGKdD), was incubated with cockroach extracts, which had been pretreated with medium, pepstatin A agarose or control agarose. As expected, trypsin cleaved the peptide substrate between Arg36 and Ser37 and produced the S37LIGKdD fragment (Figure 3); pretreatment of trypsin with pepstatin A agarose showed no effect on the cleavage (see Table E1 in this article’s Online Repository at www.jacionline.org). Both German and Oriental cockroach extracts cleaved the peptide substrate between Arg36 and Ser37, producing an S37LIGKdD fragment (Figure 3 and Table E1). Pretreatment of German and Oriental cockroach extracts with pepstatin A inhibited production of the S37LIGKdD peptide fragment by about 50~70%. Thus, these cockroach extracts likely contain pepstatin A-sensitive protease(s), which cleaves PAR-2 peptide similarly to the authentic PAR-2 ligand, trypsin. Figure 3 German cockroach extract cleaves PAR-2 peptide substrate between arginine and serine While none of the well-characterized cockroach allergens showed proteolytic activity, the midgut of cockroaches contains trypsin, chymotrypsin, subtilisin, and cysteine protease-like proteases (9). Typically, PAR-2 is cleaved and activated by serine proteases, such as trypsin (7). However, in a human epithelial cell line stimulated with German cockroach extract, the aspartate protease inhibitor, pepstatin A, and the cysteine protease inhibitor, E64, inhibited phospho-p44 MAP kinase levels, suggesting a role for cysteine proteases or aspartate proteases. Furthermore, exogenous chitinase from a bacterium, Streptomyces griseus, cleaved human PAR-2 peptide and induced a PAR-2-dependent [Ca2+]i response. These previous observations and our current findings suggest that PAR-2 recognizes both conventional trypsin-like proteases and perhaps other proteases and glycosidases derived from microbes, fungi and insects. Understanding these cockroach protease molecules and their receptors on immune cells may create novel strategies to prevent and to treat bronchial asthma.