|
|
Blood, 1 October 2007, Vol. 110, No. 7, pp. 2772-2773.
 Previous Article | Table of Contents | Next Article 
CORRESPONDENCE
Expression of granule-associated proteins in neutrophils from patients with severe congenital neutropenia
To the editor:
In a recent publication in Blood, Donini et al1 conclude that granulocyte colony-stimulating factor (G-CSF) treatment of severe congenital neutropenia (SCN) results in abnormal expression of granule-associated proteins. We have previously published this concept,2 and showed that neutrophils from patients with SCN lack the antimicrobial peptide LL-37 and have reduced levels of defensins (HNP1–3), but are functionally capable of both phagocytosis and generating oxidative radicals. Our recent results (Figure 1) differ in part to those of Donini et al, and we would like to address this discrepancy.
View larger version (69K):
[in this window]
[in a new window]
|
Figure 1. Immunoblot detection of granule proteins in neutrophils from patients with SCN. Polymorphonuclear cells (PMNs) were isolated by Dextran T-500 sedimentation (Amersham Pharmacia Biotech, Uppsala, Sweden) prior to density centrifugation using Lymphoprep (Axis-Shield, Oslo, Norway). Remaining erythrocytes were lysed by hypotonic shock, and cell purity (> 95%) was determined using light microscopy. PMNs were lysed by sonification in water that contained protease inhibitors (Complete; Roche, Indianapolis, IN) and proteins were separated with SDS-PAGE (NuPAGE 4%–12%; Invitrogen, Carlsbad, CA).3 Lysate corresponding to 4 µg protein was loaded in each lane. C indicates control participants; no. 1, the patient who underwent bone marrow transplantation; and nos. 2, 3, 7, and 15, the patients with SCN. Proteins were detected with specific antibodies against the following granule proteins: HNP1–3 (BD Biosciences Pharmingen, Palo Alto, CA), lysozyme, MPO, lactoferrin (all from DAKO A/S, Glostrup, Denmark), LL-37,2 and gelatinase (Jack Cowland, Rigshospitalet, Copenhagen, Denmark). Histone H1 (Acris Antibodies, Hiddenhausen, Germany) was used as an internal control. Bound antibody was detected with horseradish peroxidase (HRP)–conjugated antibodies (goat anti–rabbit IgG [H + L] was from BioRad Laboratories [Hercules, CA] and goat anti-mouse was from Pierce Biotechnology [Rockford, IL]), followed by chemiluminescence using SuperSignal West Dura (Pierce Biotechnology).
|
|
SCN is a heterogeneous disease that is characterized by bone marrow failure to produce normal numbers of mature neutrophils. The mechanisms behind this arrest involve apoptosis and 2 genes that are frequently mutated, HAX1 and ELA2, in autosomal recessive and autosomal dominant/sporadic SCN, respectively (reviewed in Skokowa et al4). Without correction of neutrophil levels, the patients are at risk for life-threatening infections. Despite treatment with G-CSF that normally reverses neutropenia, many patients still are at risk for infections, notably in the oral cavity.5
We have recently analyzed circulating neutrophils from 4 patients with SCN and compared with 3 control participants and 1 patient who underwent bone marrow transplantation. Lysozyme, myeloperoxidase (MPO), and lactoferrin are produced at similar levels, while neutrophils from some but not all patients with SCN have lower levels of gelatinase and HNP1–3 (Figure 1). Compared with the results of Donini et al, our patients are not deficient in lactoferrin, HNP1–3 (mature), or MPO. In addition, Donini et al noted normal levels of ELA2 mRNA, while Skokowa et al4 present patients with reduced ELA2 mRNA levels.
What could be the reason for this variation in expression, and is it of functional significance? One important issue may be the responsiveness to G-CSF treatment. Although the authors state that G-CSF reverses neutropenia (which commonly is the case), 2 of their patients in fact respond poorly to the treatment, and thus a proper increase of neutrophil protein synthesis may not occur. A second issue is the cohort chosen. It is estimated that about 60% of patients with SCN harbor mutations in ELA2.6 However, more than 40 different sites for mutations are known, and thus the impact of a specific mutation may vary.7 Donini et al investigated 3 patients with 2 different ELA2 mutations (G185R, P176fsX182), while our study included 2 patients with ELA2 mutations at other locations (L92H, C26S).3
Besides abnormal protein expression, neutrophils from patients with SCN are known to respond poorly to fMLP with diminished chemotaxis and O2– generation,8 the latter in accordance to findings by Donini et al. A previous report concludes that neutrophils from patients with SCN kill Staphylococcus aureus,9 while Donini et al reported reduced killing of Escherichia coli and Candida albicans.
Taken together, the results presented by Donini et al, interesting as they are, may be restricted to certain ELA2 phenotypes, especially G185R, and not a general phenomenon of patients with SCN. Additional clinical details of the patients presented by Donini et al would have been useful for comparison to earlier work, but also to understand the pathophysiology of SCN.
Authorship
Conflict-of-interest disclosure: The authors declare no competing financial interests.
This work was supported by the Swedish Research Council, the Swedish Society for Medical Research, The Magnus Bergwall Foundation, the Swedish Medical Society, and the Swedish Children's Cancer Foundation.
Correspondence: Mats Andersson, Department of Microbiology, Tumor and Cell Biology, Karolinska Institutet, Box 280, 17177 Stockholm, Sweden; e-mail: mats.andersson.2{at}ki.se.
Mats Andersson,
Jenny Karlsson,
Göran Carlsson, and
Katrin Pütsep
References
- Donini M, Fontana S, Savoldi G, et al. G-CSF treatment of severe congenital neutropenia reverses neutropenia but does not correct the underlying functional deficiency of the neutrophil in defending against microorganisms. Blood 2007; 109:4716–4723.[Abstract/Free Full Text]
- Putsep K, Carlsson G, Boman HG, Andersson M. Deficiency of antibacterial peptides in patients with morbus Kostmann: an observation study. Lancet 2002; 360:1144–1149.[CrossRef][Medline]
[Order article via Infotrieve]
- Karlsson J, Carlsson G, Göransdotter Ramme K, et al. Low plasma levels of the protein pro-LL-37 as an early indication of severe disease in patients with chronic neutropenia. Br J Haematol 2007; 137:166–169.[CrossRef][Medline]
[Order article via Infotrieve]
- Skokowa J, Germeshausen M, Zeidler C, Welte K. Severe congenital neutropenia: inheritance and pathophysiology. J Curr Opin Hematol 2007; 14:22–28.
- Carlsson G, Wahlin YB, Johansson A, et al. Periodontal disease in patients from the original Kostmann family with severe congenital neutropenia. J Periodontol 2006; 77:744–751.[CrossRef][Medline]
[Order article via Infotrieve]
- Bellanne-Chantelot C, Clauin S, Leblanc T, et al. Mutations in the ELA2 gene correlate with more severe expression of neutropenia: a study of 81 patients from the French Neutropenia Register. Blood 2004; 103:4119–4125.[Abstract/Free Full Text]
- Dale DC, Person RE, Bolyard AA, et al. Mutations in the gene encoding neutrophil elastase in congenital and cyclic neutropenia. Blood 2000; 96:2317–2322.[Abstract/Free Full Text]
- Elsner J, Roesler J, Emmendorffer A, Lohmann-Matthes ML, Welte K. Abnormal regulation in the signal transduction in neutrophils from patients with severe congenital neutropenia: relation of impaired mobilization of cytosolic free calcium to altered chemotaxis, superoxide anion generation and F-actin content. Exp Hematol 1993; 21:38–46.[Medline]
[Order article via Infotrieve]
- Roesler J, Emmendorffer A, Elsner J, Zeidler C, Lohmann-Matthes M, Welte K. In vitro functions of neutrophils induced by treatment with rhG-CSF in severe congenital neutropenia. Eur J Haematol 1991; 46:112–118.[Medline]
[Order article via Infotrieve]
 CiteULike  Connotea  Del.icio.us  Digg  Reddit  Technorati What's this?
Related Articles in Blood Online:
-
Response: ELA2 genotype-phenotype correlation to be expected in patients with severe congenital neutropenia
- Raffaele Badolato, Marta Donini, Gianfranco Savoldi, and Stefano Dusi
Blood 2007 110: 2773-2774.
[Full Text]
[PDF]
-
G-CSF treatment of severe congenital neutropenia reverses neutropenia but does not correct the underlying functional deficiency of the neutrophil in defending against microorganisms
- Marta Donini, Stefania Fontana, Gianfranco Savoldi, William Vermi, Laura Tassone, Francesca Gentili, Elena Zenaro, Daniela Ferrari, Lucia D. Notarangelo, Fulvio Porta, Fabio Facchetti, Luigi D. Notarangelo, Stefano Dusi, and Raffaele Badolato
Blood 2007 109: 4716-4723.
[Abstract]
[Full Text]
[PDF]
This article has been cited by other articles:
|
|
|
|
 
J. Karlsson, G. Carlsson, O. Larne, M. Andersson, and K. Putsep
Vitamin D3 induces pro-LL-37 expression in myeloid precursors from patients with severe congenital neutropenia
J. Leukoc. Biol.,
November 1, 2008;
84(5):
1279 - 1286.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
