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Eur Respir J 2005; 26:744-745
Copyright ©ERS Journals Ltd 2005

Activation of human lung mast cells by monomeric immunoglobulin E

S. Hart and I. Dransfield

MRC Centre for Inflammation Research, University of Edinburgh, Edinburgh, UK

To the Editors:

Immunoglobulin (Ig) preparations contain variable proportions of Ig aggregates 1, which behave like true immune complexes and are able to activate inflammatory cells by crosslinking Fc receptors 2. The validity of the study by Cruse et al. 3, which appeared in a previous issue of the European Respiratory Journal and showed that monomeric IgE activates human lung mast cells, requires demonstration to show that there were no IgE aggregates in the IgE preparations. The authors proposed that centrifugation at 14,000xg for 20 min would remove any aggregated IgE, but it is unlikely that this was effective. We have examined the effect of centrifugation on binding of IgG-containing immune complexes to the low-affinity IgG receptor Fc{gamma}RIIA on apoptotic neutrophils. Fluorescent immune complexes were generated by combining monoclonal mouse IgG1 anti-fetuin with fluorescein-conjugated fetuin in conditions of antigen excess, and then subjected to centrifugation at 14,000xg or 300,000xg for 20 or 60 min, respectively. Binding of immune complexes in the post-centrifugation supernatants to apoptotic human neutrophils was measured by flow cytometry as previously described 4. The results are presented in table 1Go.


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  		Table. 1— Binding of immune complexes

 
These results demonstrate that centrifugation at 14,000xg for 20 min does not effectively deplete immune complexes. However, centrifugation at 300,000xg for 60 min removes the majority of immune complex binding, but this would be inadequate for studies of cellular activation in which even immunoglobulin dimers may induce functional responses 5. In contrast to centrifugation, size exclusion chromatography would be a reliable method for purifiying monomeric immunoglobulin E for use in mast cell activation studies.

REFERENCES

  1. Herrera AM, Saunders NB, Baker JR Jr. Immunoglobulin composition of three commercially available intravenous immunoglobulin preparations. J Allergy Clin Immunol 1989;84:556–561.[Medline] [Order article via Infotrieve]
  2. McCarthy DA, Drake AF. Spectroscopic studies on IgG aggregate formation. Mol Immunol 1989;26:875–881.[Medline] [Order article via Infotrieve]
  3. Cruse G, Kaur D, Yang W, Duffy SM, Brightling CE, Bradding P. Activation of human lung mast cells by monomeric immunoglobulin E. Eur Respir J 2005;25:858–863.[Abstract/Free Full Text]
  4. Hart SP, Jackson C, Kremmel LM, et al. Specific binding of an antigen-antibody complex to apoptotic human neutrophils. Am J Pathol 2003;162:1011–1018.[Abstract/Free Full Text]
  5. Cyster JG, Williams AF. The importance of cross-linking in the homotypic aggregation of lymphocytes induced by anti-leukosialin (CD43) antibodies. Eur J Immunol 1992;22:2565–2572.[Medline] [Order article via Infotrieve]




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