To be, or not to be, an eosinophil: that is the ???

In this issue of Blood, Doyle et al provide evidence that knockout of the genes encoding the two most abundant eosinophil secondary granule proteins disrupts the normal differentiation of eosinophils from progenitors in the bone marrow, providing a novel strain of mice with a highly specific deficiency in eosinophilopoiesis and, therefore, eosinophils. This strain is likely to be used by investigators to elaborate the normal vs pathogenic roles of eosinophils in health and disease.

Expression of the secondary granule proteins major basic protein 1 (MBP-1) and eosinophil peroxidase (EPX) is required for eosinophilopoiesis in mice.

Eosinophil activities are often linked with allergic diseases such as asthma and the pathologies accompanying helminth infection. These activities have been hypothesized to be mediated, in part, by the release of cationic proteins stored in the secondary granules of these granulocytes. The majority of the proteins stored in these secondary granules (by mass) are major basic protein 1 (MBP-1) and eosinophil peroxidase (EPX). Unpredictably, a knockout approach targeting the genes encoding these proteins demonstrated that, unlike in mice containing a single deficiency of only MBP-1 or EPX, the absence of both granule proteins resulted in the near complete loss of peripheral blood eosinophils with no apparent impact on any other hematopoietic lineage. Moreover, the absence of MBP-1 and EPX promoted a concomitant loss of eosinophil lineage-committed progenitors in the marrow, identifying a specific blockade in eosinophilopoiesis as the causative event. Significantly, this blockade of eosinophilopoiesis is also observed in ex vivo cultures of marrow progenitors and is not rescued in vivo by adoptive bone marrow engraftment, suggesting a cell-autonomous defect in marrow progenitors. These observations implicate a role for granule protein gene expression as a regulator of eosinophilopoiesis and provide another strain of mice congenitally deficient of eosinophils.

Distribution of major basic protein on human airway following in vitro eosinophil incubation.

Major basic protein (MBP) released from activated eosinophils may influence airway hyperresponsiveness (AHR) by either direct effects on airway myocytes or by an indirect effect. In this study, human bronchi, freshly isolated human eosinophils, or MBP purified from human eosinophil granules were incubated for studying eosinophil infiltration and MBP localization. Eosinophils immediately adhered to intact human airway as well as to cultured human airway myocytes and epithelium. Following incubation 18-24 h, eosinophils migrated into the airway media, including the smooth muscle layer, but had no specific recruitment to airway neurons. Eosinophils released significant amounts of MBP within the airway media, including areas comprising the smooth muscle layer. Most deposits of MBP were focally discrete and restricted by immunologic detection to a maximum volume of approximately 300 microm(3) about the eosinophil. Native MBP applied exogenously was immediately deposited on the surface of the airway, but required at least 1 h to become detected within the media of the airway wall. Tissue MBP infiltration and deposition increased in a time- and concentration-dependent manner. Taken together, these findings suggest that eosinophil-derived cationic proteins may alter airway hyperresponsiveness (AHR) in vivo by an effect that is not limited to the bronchial epithelium.

Mechanisms of eosinophil major basic protein-induced hyperexcitability of vagal pulmonary chemosensitive neurons.

We have reported recently that eosinophil-derived basic proteins directly enhance the capsaicin- and electrical stimulation-evoked whole cell responses in rat pulmonary sensory neurons (19). Our present study further elucidates the mechanisms underlying the sensitization of pulmonary afferent nerves induced by these cationic proteins. Our results show that pretreatment with eosinophil major basic protein (MBP; 2 microM, 60 s) significantly enhanced the excitability of isolated rat vagal pulmonary chemosensitive neurons to acid and ATP in the current-clamp mode, but this potentiating effect was absent in the voltage-clamp recordings. The hyperexcitability induced by MBP was not prevented by the blockade of either transient receptor potential vanilloid type-1 receptor (TRPV1) selectively (inhibitor: AMG 9810; 1 microM, 2 min) or all TRPV1-4 channels (inhibitor: ruthenium red; 5 microM, 2 min). In addition, MBP also markedly potentiated the excitability of mouse pulmonary chemosensitive neurons, and no detectable difference was found between those isolated from wild-type and TRPV1 knockout mice. Furthermore, MBP pretreatment affected the decay time and recovery phase of the action potentials evoked by current injections and significantly inhibited both the sustained delayed-rectifier voltage-gated K(+) current (IK(dr)) and the A-type, fast-inactivating K(+) current (IK(a)) in these sensory neurons. In conclusion, our results indicate that the inhibition of IK(dr) and IK(a) should, at least in part, account for the hyperexcitability of pulmonary chemosensitive neurons induced by eosinophil-derived cationic proteins, whereas an interaction with TRPV1 channels does not seem to be required for the sensitizing effect of these proteins.

Major basic protein-1 promotes fibrosis of dystrophic muscle and attenuates the cellular immune response in muscular dystrophy.

The immune response to dystrophin-deficient muscle promotes the pathology of Duchenne muscular dystrophy (DMD) and the mdx mouse model of DMD. In this investigation, we find that the release of major basic protein (MBP) by eosinophils is a prominent feature of DMD and mdx dystrophy and that eosinophils lyse muscle cells in vitro by the release of MBP-1. We also show that eosinophil depletions of mdx mice by injections of anti-chemokine receptor-3 reduce muscle cell lysis, although lysis of mdx muscle membranes is not reduced by null mutation of MBP-1 in vivo. However, ablation of MBP-1 expression in mdx mice produces other effects on muscular dystrophy. First, fibrosis of muscle and hearts, a major cause of mortality in DMD, is greatly reduced by null mutation of MBP-1 in mdx mice. Furthermore, either ablation of MBP-1 or eosinophil depletion causes large increases in cytotoxic T-lymphocytes (CTLs) in mdx muscles. The increase in CTLs in MBP-1-null mice does not reflect a general shift toward a Th1 inflammatory response, because the mutation had no significant effect on the expression of interferon-gamma, inducible nitric oxide synthase or tumor necrosis factor. Rather, MBP-1 reduces the activation and proliferation of splenocytes in vitro, indicating that MBP-1 acts in a more specific immunomodulatory role to affect the inflammatory response in muscular dystrophy. Together, these findings show that eosinophil-derived MBP-1 plays a significant role in regulating muscular dystrophy by attenuating the cellular immune response and promoting tissue fibrosis that can eventually contribute to increased mortality.

Lack of eosinophil peroxidase or major basic protein impairs defense against murine filarial infection.

Eosinophils are a hallmark of allergic diseases and helminth infection, yet direct evidence for killing of helminth parasites by their toxic granule products exists only in vitro. We investigated the in vivo roles of the eosinophil granule proteins eosinophil peroxidase (EPO) and major basic protein 1 (MBP) during infection with the rodent filaria Litomosoides sigmodontis. Mice deficient for either EPO or MBP on the 129/SvJ background developed significantly higher worm burdens than wild-type mice. Furthermore, the data indicate that EPO or MBP is involved in modulating the immune response leading to altered cytokine production during infection. Thus, in the absence of MBP, mice showed increased interleukin-10 (IL-10) production after stimulation of macrophages from the thoracic cavity where the worms reside. In addition to elevated IL-10 levels, EPO(-/-) mice displayed strongly increased amounts of the Th2 cytokine IL-5 by CD4 T cells as well as a significantly higher eosinophilia. Interestingly, a reduced ability to produce IL-4 in the knockout strains could even be seen in noninfected mice, arguing for different innate propensities to react with a Th2 response in the absence of either EPO or MBP. In conclusion, both of the eosinophil granule products MBP and EPO are part of the defense mechanism against filarial parasites. These data suggest a hitherto unknown interaction between eosinophil granule proteins, defense against filarial nematodes, and cytokine responses of macrophages and CD4 T cells.

The proform of the eosinophil major basic protein binds the cell surface through a site distinct from its C-type lectin ligand-binding region.

The highly basic eosinophil major basic protein (MBP), present in the crystalloid core of eosinophil leukocyte granules, has both cytotoxic and cytostimulatory properties and is directly implicated in a number of diseases. The crystal structure of MBP resembles that of the C-type lectin (CTL) superfamily, and recent data showed that MBP binds heparan sulfate glycosaminoglycan (GAG), with the CTL ligand-binding region as the binding site. MBP is synthesized as a proform (pro-MBP) containing an acidic propiece believed to neutralize the basic MBP domain. Using flow cytometry and site-directed mutagenesis, we demonstrate here that the MBP domain of pro-MBP binds to heparan sulfate GAG on the cell surface and that this is independent of GAG covalently bound to pro-MBP. Eight basic residues located in the CTL ligand-binding region of MBP were hypothesized previously to mediate GAG binding, but we found that surface binding was not compromised by the substitution of these residues with alanine. However, the analysis of a series of mutants with surface-exposed residues substituted with alanine showed that Ser-166, Arg-168, and Arg-171 are involved in surface binding. A binding site formed by these residues is located in the MBP domain between loop 1 and beta-strand 5, outside the CTL ligand-binding region. The binding of a cell-surface heparan sulfate proteoglycan may be important in MBP action, and our findings suggest that two regions shown previously to contain the cytotoxic and cytostimulatory properties of MBP are accessible for ligand interaction in cell surface-bound MBP.

Eosinophils, but not eosinophil peroxidase or major basic protein, are important for host protection in experimental Brugia pahangi infection.

The attenuation of eosinophilia by the administration of monoclonal antibodies to CCR3 consistently correlates with impairment in worm elimination following primary intraperitoneal Brugia pahangi infections in mice. Host protection was unimpaired in mice deficient in eosinophil peroxidase (EPO) or major basic protein 1 (MBP-1), suggesting that eosinophils are essential in host protection but that neither EPO nor MBP-1 alone is.

Eosinophils alter colonic epithelial barrier function: role for major basic protein.

Mucosal eosinophils increase in a number of gastrointestinal diseases that are often associated with altered epithelial barrier function, including food allergic enteropathies and inflammatory bowel diseases. Although eosinophils are known to secrete biologically active mediators including granule proteins, their role in gastrointestinal diseases is uncertain. The aim of this study was to determine the impact of eosinophils on intestinal barrier function. Epithelial barrier function was determined in a coculture of eosinophils and T84 epithelial cells and in a murine model of T helper (Th) type 2-mediated colitis. Coculture conditions resulted in decreased transepithelial resistance (TER) and increased transepithelial flux. Cell-free coculture supernatants contained a > or =5-kDa soluble factor that also diminished TER; these supernatants contained the eosinophil-granule proteins major basic protein (MBP) and eosinophil-derived neurotoxin (EDN). T84 barrier function decreased significantly when basolateral surfaces were exposed to native human MBP but not EDN. Additional studies identified downregulation of the tight junctional molecule occludin as at least one mechanism for MBP action. MBP-null mice were protected from inflammation associated with oxazolone colitis compared with wild-type mice. In conclusion, MBP decreases epithelial barrier function and in this manner contributes to the pathogenesis of inflammatory bowel diseases.

Proteinase inhibition by proform of eosinophil major basic protein (pro-MBP) is a multistep process of intra- and intermolecular disulfide rearrangements.

The metzincin metalloproteinase pregnancy-associated plasma protein A (PAPP-A, pappalysin-1) promotes cell growth by the cleavage of insulin-like growth factor-binding proteins-4 and -5, causing the release of bound insulin-like growth factors. The proteolytic activity of PAPP-A is inhibited by the proform of eosinophil major basic protein (pro-MBP), which forms a covalent 2:2 proteinase-inhibitor complex based on disulfide bonds. To understand the process of complex formation, we determined the status of cysteine residues in both of the uncomplexed molecules. A comparison of the disulfide structure of the reactants with the known disulfide structure of the PAPP-A.pro-MBP complex reveals that six cysteine residues of the pro-MBP subunit (Cys-51, Cys-89, Cys-104, Cys-107, Cys-128, and Cys-169) and two cysteine residues of the PAPP-A subunit (Cys-381 and Cys-652) change their status from the uncomplexed to the complexed states. Upon complex formation, three disulfide bonds of pro-MBP, which connect the acidic propiece with the basic, mature portion, are disrupted. In the PAPP-A.pro-MBP complex, two of these form the basis of both two interchain disulfide bonds between the PAPP-A and the pro-MBP subunits and two disulfide bonds responsible for pro-MBP dimerization, respectively. Based on the status of the reactants, we investigated the role of individual cysteine residues upon complex formation by mutagenesis of specific cysteine residues of both subunits. Our findings allow us to depict a hypothetical model of how the PAPPA.pro-MBP complex is formed. In addition, we have demonstrated that complex formation is greatly enhanced by the addition of micromolar concentrations of reductants. It is therefore possible that the activity in vivo of PAPP-A is controlled by the redox potential, and it is further tempting to speculate that such mechanism operates under pathological conditions of altered redox potential.