Monocytes are selectively recruited to different tissues in response to either infection, injury (9
), or tissue-specific recruitment factors (30
). Upon entry into a tissue, monocytes differentiate into macrophages, which display a phenotype regulated by the local tissue environment (27
). Adoptive transfer of C2D macrophages into mice provided enhanced resistance to infection (33
). However, it was not clear what happened to those macrophages in the recipient. In this study, we characterized the response of the macrophage cell line C2D to the in vivo microenvironment of the peritoneal cavity in an effort to understand the underlying changes that lead to the protective response. The peritoneal cavity offered a convenient and sterile site (12
) to explore the complexity of the in vivo environment and its effects on the C2D macrophage phenotype. The adoptive transfer of C2D macrophage cells allows long-term survival in vivo in the absence of detrimental pathology (33
). This is important for studying the effects of the local, uninflamed microenvironment.
C2D macrophage cells responded to in vivo signals (Fig. ). When grown in vitro, C2D macrophage cells have an immature phenotype based on their low-level expression of the mature macrophage markers F4/80 and CD11b (38
), their mid-level expression of Mac-2 (42
), and their low-level production of the macrophage cytokine TNF-α. Within the peritoneal cavity, C2D macrophage cells were induced to differentiate from immature macrophages to mature macrophages. They expressed high levels of F4/80, CD11b, Mac-2, and c-fms, (37
). The increase in Ly-6C after adoptive transfer, which can be induced only on immature cells, also confirms the maturation of the C2D macrophages (36
). The expression of F4/80 and CD11b and the up-regulation of TNF-α activity by C2D macrophage cells (Fig. ) were also consistent with the phenotype of differentiated macrophages (49
). In fact, the ability of C2D macrophage cells to make TNF-α in vivo supports the observation of earlier TNF-α production in lungs after P. pneumotropica
infection in mice adoptively transferred with C2D macrophage cells, which leads to an altered early inflammatory response (3
FIG. 8. Antigen expression during C2D macrophage cell differentiation. The figure is based on data from references 36 to 39. *, percent expression on C2D macrophage cells in vitro; **, percent expression on C2D macrophage cells after in (more ...)
We expected there to be some influence of mouse strain on the phenotype of adoptively transferred C2D macrophage cells. The observation that there was no strain dependence suggests that TLR4 and MHC-II genes (or molecules) are not involved in macrophage differentiation and that nonpolymorphic factors independent of these genes are present in all the mouse strains tested. One factor that may play an important role in C2D macrophage function is integrin and/or other adhesion molecules. Knockout mice for some of these molecules exist (e.g., Mac-1 [9
] or P/L/E-selectin [14
]). Future studies on how C2D macrophage cells traffic in the in vivo environment in these mice may provide insight as to which factors influence the phenotype of C2D macrophage cells found in the spleen, adipose tissue, and lymph nodes.
Although IFN-γ had little effect on many macrophage phenotypic markers in this study, we did see an increase in Gr-1 and a decrease in c-fms expression. The lack of a change in so many molecules was somewhat surprising. IFN-γ has profound effects on macrophage function and activation (2
) and influences monocyte maturation (17
). The increase in Gr-1 may reflect activation, as macrophage granularity tends to positively correlate with activation (10
). IFN-γ is also required for the generation of inhibitory macrophages capable of suppressing T-cell proliferation, which are identified in part by their expression of Gr-1 (24
). The decrease in c-fms expression was somewhat surprising because c-fms expression has been shown to be relatively stable, expressed under both steady-state and inflammatory conditions (12
). c-fms mRNA transcripts were also unaffected by IFN-γ treatment (32
). A possible explanation for the decrease in c-fms surface expression may be increased internalization after IFN-γ stimulation (17
). The surface expression of c-fms has also been noted to decrease (while mRNA levels remain increased) on microglial cells within the lesions of multiple sclerosis patients (70
). These lesions are characterized by inflammation and tissue destruction (45
). As an alternative measure of activation, we noted an increase in nonspecific Ig binding while doing flow cytometry measurements (data not shown), which is indicative of increased surface expression of Fc receptors caused by IFN-γ (15
C2D macrophage cells responded to IFN-γ in vivo in a similar fashion in all three mouse strains. There is a tight interaction between the signaling pathways of TLR4 and IFN-γ (11
), and it was expected that there would be a significant impact on macrophage phenotype in the absence of TLR4. However, C2D macrophage cells transcribe their own TLR4 if it is needed for activation and thus may respond independently of host expression. The presence of MHC-II may also be irrelevant for activation by IFN-γ. Alternatively, we introduced IFN-γ directly to the C2D macrophage cells within the peritoneal cavity, and therefore, the cells were stimulated directly. It is possible that if stimulated indirectly (i.v.), the agonist would stimulate immunological components of the host and thus may impact the C2D macrophage cells in a more host-dependent manner.
The ability of C2D macrophage cells to traffic and be retained in vivo demonstrates that macrophages adapted to long-term in vitro culture can be reintroduced in vivo, traffic to distant organs, and reside in the host for longer than 2 months without causing pathology. Although other researchers have adoptively transferred macrophage cell lines to restore host immunity, few studies have attempted to maintain the cells in vivo for extended lengths of time. Wiltrout et al. previously found that the WEHI-3 and RAW264 macrophage cell lines injected i.v. were quickly removed over time (72
). Nishihara et al. found that transfected J774A.1 macrophages functioned up to 20 days following intratumor injection (50
). We detected cells from the C2D macrophage cell line up to 62 days posttransfer, suggesting that they persist and behave in a manner similar to that of resident macrophages and that they may be maintained indefinitely (we did not assay for the presence of cells after 62 days). This novel difference in longevity may be due to subtle differences in cell line characteristics including differentiation state, surface antigen expression (i.e., the lack of MHC-II), and cytokine production.
The detection of cells derived from the C2D macrophage cell line in vivo using RT-PCR was complemented by the identification of whole cells using confocal microscopy and fluorescence-activated cell sorter analysis. We identified C2D macrophage cells in various tissues including bone marrow, spleen, lungs, pancreatic lymph nodes, and perinodal adipose tissue. Their trafficking is consistent with the localization of macrophages throughout the body (28
). Brunstetter et al. previously analyzed the migration of adoptively transferred primary bone marrow cells and determined that it took 8 weeks for the immature cells to appear in the alveolar compartment of normal mice (7
). Slightly more mature Ly-6C-positive mononuclear cells traffic to the spleen, lung, and liver by approximately 3 days posttransfer (37
), while adoptively transferred peritoneal macrophages migrate to the spleen and lymph nodes by 4 h posttransfer and remain up to 6 days (9
). Thus, the differentiated phenotype of C2D macrophage cells facilitates their dispersal to peripheral tissue and could prove useful in further investigating the effects of microenvironmental influences on macrophage heterogeneity in future studies.
It is clear that C2D macrophage cells divided in vivo. Cells labeled with CFDA SE were difficult to detect after 3 to 4 days in vivo, indicating that cell division was diluting out the label. Their detection 2 months after injection by RT-PCR could be due to long-lived cells from the original transplant or cells that have divided locally. If the latter possibility is true, C2D cells divided without being tumorigenic (3
), and there was no evidence of neoplasia at necropsy or by histology at 1 or 2 months post-adoptive transfer. Local macrophage cell division is not unprecedented. In a quantitative evaluation of macrophage kinetics, 30% of host alveolar macrophages arose from a local population of dividing mononuclear phagocytes (6
Differentiation of bone marrow cells in the in vitro environment using various cytokines has long since been established and used to develop a number of cell lines (35
). M-CSF is used to differentiate bone marrow cells into macrophages (40
), whereas GM-CSF and IL-4 are used to differentiate bone marrow cells into dendritic cells (69
). Bone marrow-derived macrophages have been shown to express Mac-1, Mac-2, and F4/80 (40
), while the addition of TNF-α to bone marrow results in a myeloid-dendritic cell phenotype, expressing both CD11b and CD11c (69
). The treatment of C2D macrophage cells in vitro with either M-CSF or GM-CSF and IL-4 had little to no effect on their phenotype, unlike the phenotype of treated bone marrow cells. As an alternative method of altering the phenotype of C2D macrophage cells in vitro, we treated them with dexamethasone and insulin (data not shown). The addition of dexamethasone and insulin, which causes the differentiation of 3T3-L1 fibroblasts into adipocytes (26
), has been shown by our group (L. Xie and S. K. Chapes, unpublished data) to increase the production of IL-6 by C2D macrophage cells in vitro. However, we saw no alteration of the phenotype of C2D macrophage cells after treatment with dexamethasone and insulin.
In summary, our data demonstrate that the peritoneal cavity altered the surface expression of C2D macrophage cells, causing them to differentiate from immature cells to mature macrophages. These changes were dependent on complex signals from the host (64
) that were not present in vitro, as demonstrated by the insignificant change in the phenotype of C2D macrophage cells after the addition of differentiation factors in vitro. Future studies will include examining the role of extracellular matrices in the differentiation of C2D macrophage cells.