Our findings demonstrate that MHC Class II can migrate and localize to DRMs in human eosinophils in a functionally significant manner. The association of MHC Class II and the tetraspanin CD9 with DRMs in eosinophils adds to the evidence that eosinophils have important professional APC functions. Although there is accumulating evidence supporting eosinophils as important and potent APCs, a gap exists in the understanding of the structural and organizational aspects of the antigen presentation complex in eosinophils. Distinct parallels can be drawn between the present study of human eosinophils and prior studies of DCs and other professional APCs, in which both tetraspanin molecules and DRMs likely play a pivotal role in the organization of the antigen presentation complex.
The tetraspanins are a group of proteins belonging to the transmembrane-4 superfamily that have major function in the organization of protein complexes in cell membranes (28
). Unternaehrer and colleagues demonstrated in mouse DCs that the tetraspanin CD9 associates with MHC Class II molecules in the plasma membrane (18
). Their data showed that CD9 mediates the association of heterologous MHC Class II molecules, suggesting that CD9 plays a key role in organizing antigen-presenting molecules in a manner that allows for more efficient APC function. They also found that the CD9 expression on the plasma membrane of murine DCs was high, whereas it was low on “non-professional” APCs such as B blasts. We have demonstrated here that surface expression of CD9 is comparable between human eosinophils and monocyte-derived DCs, further supporting eosinophils as professional APCs. As seen in previous studies, CD9 is abundant on the cell surface of eosinophils to such an extent as to be considered a marker for eosinophils (13
). Here, we extend these studies showing, for the first time, the ultrastructural immunolocalization of CD9 in human eosinophils. Our EM data confirm the substantial localization of CD9 at the cell surface and the presence of CD9 clusters at areas of the plasma membrane (). Our findings strongly suggest that a subset of CD9 and HLA-DR are associated in DRMs on the surface of human eosinophils. We have found that HLA-DR and CD9 not only colocalize with one another but also colocalize with the DRM marker CTB. In addition, disruption of DRMs leads to marked decrease in expression of HLA-DR and CD9 on the surface of human eosinophils. Cholesterol depletion with MβCD does not cause a reduction in detectable GM1 by flow cytometry on the surface of eosinophils, consistent with preferential removal of DRM-associated proteins compared with gangliosides. Although patterns of GM1 expression with DRM disruption have not been studied in eosinophils, changes in GM1 distribution rather than a decrease in total GM1 content may explain this differential effect of MβCD. A prior report has demonstrated changes in GM1 distribution with MβCD treatment in murine fibroblasts (29
We have biochemically demonstrated that HLA-DR and CD9 are present in DRM fractions of human eosinophil lysates subjected to sucrose density gradient fractionation. Particularly noteworthy from a biochemical perspective are our findings regarding CD9. CD9 and other tetraspanins have been shown to associate with cholesterol and other membrane lipids in DRMs distinct from lipid rafts known as tetraspanin-enriched microdomains (TEMs) (30
). However, unlike lipid rafts, TEMs generally appear to be soluble in stronger detergents, such as Triton X-100 (28
). Our data suggest that CD9 in human eosinophils is present in lipid rafts, rather than only in TEMs, given the abundant multimeric CD9 found to have floated upward into the detergent-resistant area with sucrose density gradient fractionation of Triton X-100 eosinophil lysates ().
The present study investigated the necessity of HLA-DR–containing DRM integrity to superantigen-mediated eosinophil APC function. The role of DRMs in the organization of antigen-presenting molecules in eosinophils has been unknown to this point. In fact, the relevance of DRMs in eosinophil biology in general has not been well characterized. To our knowledge, only one study has examined DRMs in human eosinophils (12
). DRMs have previously been demonstrated to have an important role in the organization and activity of MHC Class II molecules in a variety of other APCs (9
). MHC Class II in murine B cells has been observed in DRMs; in these cells, disruption of DRMs with MβCD inhibited their ability to present antigen (10
). In human monocyte–derived DCs, HLA-DR coaggregates with the DRM markers CD59 and GM1-ganglioside when cross-linked, and disruption of DRMs biochemically eliminates DRMs from the DC-T cell synapse and inhibits T-cell activation by the DCs (11
). We functionally demonstrate that eosinophils loaded with the superantigen SEA are able to stimulate CD4+
T cells in a DRM-dependent fashion. We did so by measuring three different indicators of the activation state of T cells: expression of the early activation marker CD69 at 24 hours, production of cytokines by T cells at 48 hours, and proliferation of T cells by CFSE dilution at 96 hours. The ability of human eosinophils to stimulate T cells by these metrics is a novel finding.
Up until this point, the structure of the antigen-presentation complex on eosinophils has been an unexplored topic, particularly given that eosinophils acting as professional APCs is a relatively recently described phenomenon. It is known that APCs only require a small number of MHC-peptide complexes to activate T cells (37
). Given that human eosinophils are efficient at antigen-presentation despite lower cell surface expression of HLA-DR than other professional APCs (6
), one could speculate that the potential organization of MHC Class II into DRMs on eosinophils may help explain their relative efficiency.
Also currently unresolved is the true contribution of eosinophil APC function in vivo
. Our group has previously demonstrated that eosinophils harvested from IL-5 transgenic mice and loaded with OVA traffic to paratracheal lymph nodes after intratracheal instillation into wild-type mice, where they are able to stimulate OVA-specific CD4+
T cells to express CD69, proliferate, and secrete IL-4 (7
). This observation, in concert with the present findings that human eosinophils have functionally relevant grouping of MHC Class II with CD9 in DRMs, suggests that eosinophil APC function has in vivo
physiologic, nonredundant significance.
Characterization of important immunoregulatory roles of eosinophils, including antigen presentation, has lagged behind that of other immune cell types. This lag may be in part responsible for an incomplete understanding of the pathobiology of asthma and other allergic inflammatory processes. Further investigation of the immunobiology of eosinophils has taken on increased concrete clinical importance given recent findings regarding the efficacy of eosinophil-depletion by anti-IL5 mAb in selected patients with asthma as well as their conspicuous presence in eosinophilic pulmonary disorders such as Churg-Strauss syndrome and eosinophilic pneumonia (39
). This study adds to the ever-increasing store of evidence that eosinophils are significantly more complex and multifaceted than previously appreciated.