We used mass spectrometry to provide a global view of the proteomes of macrophages and DCs generated in vitro with M-CSF or GM-CSF. Using stringent dual statistical criteria, we identified 106 proteins that were enriched in the membranes of the different cell types generated in vitro. We also identified core sets of proteins that distinguished all the macrophage types and DCs from each other ().
Plasma membrane protein signatures of myeloid cells identify unique cell functions.
Our data suggest that proteomics can distinguish macrophage classes from each other and from DCs. They are also consistent with the view that M-CSF and GM-CSF are major determinants of the polarization/differentiation of myeloid cells in vivo
. Moreover, they provide direct in vivo
evidence that GM-CSF is a major phenotypic determinant of peritoneal myeloid cells of mice challenged with thioglycolate, a classic model of sterile inflammation.
Certain of the proteins we identified are widely used as markers of cell phenotype in vivo
. For example F4/80 is generally regarded as a marker for macrophages 
, and our proteomic analyses detected much higher levels of F4/80 in the plasma membrane of macrophages than in that of DCs. We also found that some widely used markers are not specific. One striking example is CD11c – often used as a DC marker. Mass spectrometry and immunofluorescence detected high levels of CD11c in the plasma membrane of M2 macrophages. Indeed, recent findings demonstrate that CD11c fails to discriminate between macrophage and DC populations in vivo
. However, most of the proteins that were enriched in the membranes of the various cell types have not been described previously.
Because plasma membrane proteins are central to the critical functions of macrophages and DCs, our findings suggest that unique plasma membrane protein expression patterns not only interrogate the phenotypes of myeloid cells but also form an important basis for the cells' distinct functional properties. Thus, over 50% of the plasma membrane proteins we identified were selectively expressed by one of the four myeloid cell types, supporting the conclusion that polarized macrophages and DCs fulfill distinct biological roles ().
, the phenotypes of macrophages and DCs are regulated by complex, dynamic tissue environments, making it unlikely that a limited number of markers can define myeloid cell heterogeneity. Indeed, it is generally accepted that the utility of widely used myeloid markers such as F4/80, CD11b, and CD11c is highly tissue-dependent 
. Proteomic signatures, on the other hand, should be more effective tools for defining cell phenotype because patterns of protein expression are more resistant than single measurements to the highly complex and variable environments macrophages and DCs encounter in vivo
The power of using protein expression patterns to establish myeloid cell phenotypes is highlighted by the remarkable similarities we observed between the plasma membrane proteome of DCs generated with GM-CSF and that of eMPCs isolated from the inflamed peritoneum of mice. Similar findings were obtained when BmDCs and eMPCs were clustered according to gene expression patterns obtained from a meta-analysis of in vitro
and in vivo
generated myeloid cells 
. We also found that markedly fewer ePCs accumulate in GM-CSF-deficient (Csf2
−/−) mice. Functional characterization showed that cells isolated from Csf2
−/− mice were less able than those from wild-type mice to cross-present antigen (a function classically ascribed to DCs) and more able to phagocytose bacteria (a function classically ascribed to macrophages).
These observations strongly suggest that correlates of the myeloid DCs generated with GM-CSF in vitro
also exist in vivo
. Moreover, they are consistent with previous studies showing that i
) thioglycolate induces peritoneal myeloid cells to express and secrete GM-CSF 
) thioglycolate-elicited peritoneal myeloid cells emigrate to draining lymph nodes as inflammation resolves—a classic property of DCs 
, and iii
) thioglycolate-elicited peritoneal cells can present antigens and stimulate T cell proliferation 
Our proteomic analyses, together with functional analyses of macrophages and DCs by many investigators 
, depict a scenario in which M-CSF and GM-CSF support highly polarized protein expression patterns that influence diverse biological endpoints ranging from antigen presentation to cell motility, phagocytosis, generation of reactive oxygen species, fatty acid oxidation, and inflammation. Moreover, previous studies have validated many of the functional differences predicted by our proteomic data. For example, NADPH oxidase, a protein over-expressed by BmDCs, has been assigned a key role in antigen presentation by DCs in vivo
. Importantly, changes in the tissue milieu would elicit corresponding changes in resident and recruited myeloid cells. Consequently, cell types with customized patterns of protein expression (and hence function) would be generated, and such cells would be well-suited to meet the dynamic demands of the local environment.
Collectively, our observations provide a rich proteomic framework that should help investigators identify specific populations of macrophages and DCs in tissue so they can correlate functions with the correct cellular phenotypes. By using patterns of protein expression specific to each type of myeloid cell, it will now be possible to more confidently extrapolate data from in vitro experiments to more complex in vivo situations. As myeloid-derived cells are implicated in autoimmune diseases, cancer, infection, and many other conditions, our membrane proteome signatures should help investigators identify the specific populations that are central to the pathogenesis of those disorders.