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Originally published online as doi:10.1189/jlb.0607365 on December 6, 2007

Published online before print December 6, 2007
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(Journal of Leukocyte Biology. 2008;83:602-609.)
© 2008 by Society for Leukocyte Biology

Functions of C2D macrophage cells after adoptive transfer

Betsey E. Potts and Stephen K. Chapes1

Division of Biology, Kansas State University, Manhattan, Kansas, USA

1Correspondence: Division of Biology, Kansas State University, 116 Ackert Hall, Manhattan, KS 66506, USA. E-mail: skcbiol{at}ksu.edu


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ABSTRACT
 
Macrophage function depends on their in situ location. To test this hypothesis, we examined functional changes of the C2D macrophage cell line after adoptive transfer. In vitro, C2D macrophages reside early in the macrophage lineage and show little functional activity. After in vivo i.p. culture, C2D macrophage cells switch their cytokine/chemokine profile from primarily Th2 cytokines produced in vitro to a Th1 profile including MIP-1{alpha}, IL-6, and TNF-{alpha}. The in vivo environment also caused C2D macrophage cells to become more phagocytic than their in vitro counterparts. These data indicate that C2D macrophage cells exhibit distinct functions because of in vivo signals that are absent during in vitro culture.

Key Words: peritoneal cavity • microenvironment • phagocytosis • cytokines


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INTRODUCTION
 
We described previously the C2D macrophage cell line that maintained some macrophage properties including the expression of TLR4. These macrophage cells only secreted a limited number of cytokines such as IL-6 after stimulation with LPS [1 ]. Interestingly, adoptive transfer of C2D macrophage cells protected recipient-immunocompromised mice from developing pneumonia after experimental challenge with Pasteurella pneumotropica [2 ]. The mechanism for the successful adoptive transfer and protection of mice with C2D macrophage cells was unclear. Macrophage cell lines offer a novel approach to cell therapy [3 , 4 ]. Macrophages migrate to specific tissues in response to chemokine and cytokine stimuli, which make them attractive candidates for targeting host tissues with cell therapy. However, little is known about the effects of the in vivo environment on the cellular function of reintroduced in vitro-developed cell lines. Certain stimuli may induce selective secretion of one cytokine and inhibit the secretion of another. For example, macrophages stimulated with IFN-{gamma} and LPS in vitro secrete primarily proinflammatory cytokines including TNF-{alpha} and IL-6 [5 , 6 ], and macrophages stimulated with glucocorticoids tend to secrete primarily anti-inflammatory cytokines IL-4 and IL-13 [6 ]. In vivo, splenic red pulp macrophages are specialized in the uptake or phagocytosis of erythrocytes, which is important in maintaining erythrocyte homeostasis as well as in the recycling of iron [7 ], and marginal zone metallophilic macrophages, which form the inner border of the marginal zone, exhibit poor phagocytic abilities [8 ].

Our group has demonstrated that in the in vitro environment, C2D macrophage cells reside as immature macrophages, expressing moderate levels of Ly-6C and galactin-3 (Mac-2), but little F4/80 and CD11b [9 ]. However, the in vivo environment of the peritoneal cavity causes the cells to become mature macrophages, expressing high levels of F4/80, CD11b, and c-fms [9 ]. In addition to the change in phenotype, we proposed that macrophage function may be altered by the tissue microenvironment. To test this hypothesis, we assessed the function of the established macrophage cell line, C2D, after adoptive transfer to the peritoneal cavity. Here, we demonstrate that the in vivo environment of the peritoneal cavity caused C2D macrophage cells to change their functional abilities; adoptive transfer altered their cytokine/chemokine expression and increased their phagocytic activity.


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MATERIALS AND METHODS
 
Mouse strains
C57BL/6J (B6, MHCII+/+, Tlr4Lps-n) mice were originally obtained from the Jackson Laboratory (Bar Harbor, ME, USA) and were used as wild-type controls. C57BL10/ScN (B10, MHCII+/+, Tlr4Lps-del) mice were obtained from the animal resource facility at the National Institutes of Health (NIH; Bethesda, MD, USA). C2D (B6.129-AbbtmlN5F20, MHCII–/–, TLR4Lps-del) mice are on the B6 background but lack functional MHCII genes as a result of a natural deletion of the IE{alpha} gene and a targeted deletion of the IAβ gene [10 ]. B6, B10, and C2D (Table 1 ) mice were used between 6 and 10 weeks of age. A breeding pair of A2T (Table 1) heterozygote mice [11 ] was obtained from Dr. Victor Engelhard (University of Virginia, Charlottesville, VA, USA). These mice were crossed, and HLA-A2+ F2 progeny were backcrossed to A2T-negative mice to verify homozygosity of the HLA-A2 gene in the A2T mice. Mice homozygous for the HLA-A2 gene have been maintained by brother-sister mating of homozygous mice in the animal facility in the Division of Biology, Kansas State University (Manhattan, KS, USA), since 2001. All mice listed served as recipients in adoptive-transfer experiments. These mouse strains are syngeneic on the C57BL (H-2b) background and will accept graft transfers of C2D cells. All mice were bred in the rodent facility of the Division of Biology at Kansas State University. All animal experiments were approved by the Institutional Animal Care and Use Committee.


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  		Table 1. Mouse Strains

C2D macrophage cell line
The C2D macrophage cell line was created as described by our group [12 ]. These cells were derived from C2D murine bone marrow and selected in the presence of M-CSF. These cells have the MHCII–/– and Tlr4Lps-n genotype and are histocompatible with mice of the H-2b haplotype. C2D cells were grown in DMEM with 4% serum (DMEM-4), supplemented with 0.3% glutamax and 10% Opti-MEM in 100- and/or 150-mm tissue-culture plates.

Adoptive transfer of labeled cells
C2D macrophages were dispersed with 0.25% trypsin with 0.02% EDTA (TEDTA) and resuspended in DMEM-4. Cells were centrifuged at 350 g for 7 min and resuspended in warmed (37°C), sterile PBS at a concentration of 1.5 x 106 cells per ml and stained with carboxyfluorescein diacetate, succinimidyl ester (CFDA SE; Molecular Probes, Eugene, OR, USA; catalog #C1157), according to the manufacturer’s protocol. Briefly, cells were incubated with 22 µM of the probe solution at 37°C for 15 min and then centrifuged at 350 g for 7 min, resuspended in fresh, prewarmed PBS, and incubated at 37°C for an additional 20 min. After the second incubation, cells were washed two times with DMEM-4 and resuspended at a concentration of 2 x 107 cells per ml in sterile PBS containing 50 U per ml heparin (h/PBS; Elkins-Sinn, Inc., Cherry Hill, NJ, USA). The cell suspension (1 ml) was injected i.p. per mouse. CFDA SE C2D macrophages were collected from B6, B10, and C2D mice by peritoneal lavage, 1–3 days post-transfer. Most of the variation in recovery was a result of mouse size and technical issues, such as leaking of peritoneal contents during washing, and not a result of mouse strain. On average, 45% of the labeled C2D macrophage cells were recoverable 24 h after injection, 22% 72 h after injection, and 10% 1 week after injection. Therefore, we limited our analyses to 1–3 days after adoptive transfer to maximize the recovery of the labeled C2D macrophage cells.

FACS analysis
CFDA SE-labeled C2D macrophages were recovered from B6, B10, and C2D mice by peritoneal lavage, 1–3 days post-transfer with 24 ml ice-cold, sterile PBS after anesthetizing the mice with Halothane (Halocarbon, River Edge, NJ, USA) and euthanizing them by cervical dislocation. Samples were analyzed using a FACSCalibur analytical flow cytometer (Becton Dickinson, San Jose, CA, USA), 10,000–20,000 events measured for each sample. Data analysis was performed with the PC-compatible WinList software (Verity Software House, Topsham, ME, USA). Cell sorting was performed with a FACSVantage SE cell sorter (Becton Dickinson) using specimen optimization and calibration techniques, according to the manufacturer’s recommendations. Cells were sorted at a rate of 12,000 cells/s, and ~1 million cells were collected per group to minimize cell stress and damage. Cells were sorted based on CFDA SE expression, with the lowest 10% of the positive cells not selected.

Cytokine and chemokine secretion
Sorted cells were resuspended DMEM-4 containing 0.1 µg per ml gentamycin (Atlanta Biologicals, Lawrenceville, GA, USA), counted, and cultured overnight at a concentration 2 x 105 cells per 500 µl in a 24-well plate. Supernatants were then collected, centrifuged at 350 g for 5 min to remove cell debris, and frozen at –80°C until they were analyzed. Cytokine concentrations were determined using a mouse cytokine/chemokine LINCOplex (Millipore, Billerica, MA, USA; catalog #MCYTO-70K-PMX) array, according to the manufacturer’s instructions, and analyzed using the Luminex100 system.

RT-PCR
Total RNA was extracted from cells of the various treatment groups using TRI Reagent, according to the manufacturer’s instructions (Molecular Research Center, Inc., Cincinnati, OH, USA). RNA sample concentrations were determined spectrophotometrically (NanoDrop Technologies, Wilmington, DE, USA), and all samples were diluted to an optimal concentration of 100 ng per µl and a total of 500 ng per sample was used. cDNA was amplified using specific primers listed in Table 2 . Densitometric comparisons between cytokine and β-actin DNA were performed on ethidium bromide-stained 2% agarose gels using AlphaImager software (Alpha Innotech, San Leandro, CA, USA).


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  		Table 2. Primers Used for RT-PCR

Fluoresceination of Propionibacterium acnes and collection of neutrophils
Tetramethylrhodamine-5-isothiocyanate (TRITC; Sigma-Aldrich, St. Louis, MO, USA) was conjugated to heat-killed P. acnes as described previously [13 ]. Briefly, 2 ml heat-killed P. acnes (stock 7 mg/ml) was centrifuged, supernatant was decanted, and 130 µl TRITC (stock 5 mg/ml) was added and mixed thoroughly. This solution was then incubated in the dark at room temperature for 1 h. After incubation, the P. acnes-TRITC solution was washed 3x with cold PBS and resuspended in PBS at 90% of original volume. Two hundred microliters were injected per mouse with this solution. Five hours later, peritoneal cavities were washed to collect polymorphonuclear neutrophils (PMNs), of which 99% contained TRITC-labeled bacteria. The cells collected were centrifuged and resuspended in PBS, and 1 x 107 PMNs per mouse were reinjected i.p. into mice containing CFDA SE-labeled C2D cells.

Phagocytosis
In vivo
Two methods were used to assess the phagocytic activity of C2D macrophages, which effectively phagocytose neutrophils as a part of the inflammatory response. Therefore, to assess this macrophage process, we analyzed phagocytosis of TRITC-fluorescent PMNs (see above). Phagocytosis was also assessed using PE-conjugated beads (0.86 µM, Duke Scientific, Fremont, CA, USA; catalog #R900) to assess direct particle uptake. Control mice were injected with 200 µl sterile PBS. TRITC-PMNs (1x107), 300 µl beads (~0.5 PMN/C2D macrophage cell or 15 ul beads per 1x106 C2D macrophage cells), or PBS were injected i.p. into mice containing CFDA SE-labeled C2D cells on Days 1, 2, and 3 post-CFDA SE C2D cell injection. Four hours after PMN or bead injection, peritoneal cavities were washed with cold h/PBS (25 U per ml). Cells were centrifuged, washed once with TEDTA to remove all PMNs attached to the cell surface, and then resuspended in 2% formalin/PBS and analyzed by flow cytometry. Unphagocytosed PMN or beads were identified by forward-scatter (FSC) versus side-scatter (SSC) plots, and the percentage of phagocytic cells was determined from the percentage of total cells that were gated as large, granular, double-positive cells.

In vitro
C2D cells were dispersed with TEDTA, replated at a density of 1 x 106 cells per well in a 12-well plate (22.2 mm-diameter wells). Cells were allowed to adhere for 2 h. Attached control cells were placed at 4°C for 15 min before beginning the phagocytosis assay. After cold incubation, 20 µg cytochalasin D (which disrupts actin polymerization and thus, inhibits phagocytosis) and 15 µl fluorescent beads or 5 x 105 TRITC-PMNs (~0.5 PMN/C2D macrophage cell or 15 ul beads per 1x106 C2D macrophage cell) were added to control cells and kept at 4°C for 4 h. For the experimental cells, 15 µl fluorescent beads or 5 x 105 TRITC-PMNs were added and incubated at 37°C for 4 h. After incubation, cells were dispersed with TEDTA to detach them and remove any beads or neutrophils from the cell surface (not phagocytosed), washed 2x with PBS, and then resuspended in 2% formalin/PBS and analyzed by flow cytometry. To assure that only phagocytosis was analyzed, free neutrophils and beads were gated out using FSC versus SSC. Percent phagocytosis was calculated by measuring percent-positive phagocytic cells (with beads or PMNs) incubated at 37°C minus percent-positive phagocytic cells (with beads or PMNs) incubated at 4°C in the presence of cytochalasin D.

Sorting
To detect the presence of IAβ or HLA-A2 RNA in C2D macrophage cells, B6 PMNs (IAβ+) or A2T PMNs (HLA-A2+) were collected using TRITC-labeled P. acnes as described above. CFDA SE-labeled C2D cells were adoptively transferred to C2D mice, and 1 day later, 1 x 107 B6 or A2T PMNs were injected i.p. Four hours after PMN injection, peritoneal cavities were washed with cold h/PBS (25 U/ml). Cells were centrifuged and resuspended in 5% FBS in PBS at a concentration of 2 x 107 cells per ml and sorted as described previously on the basis of CFDA SE-positive and TRITC-positive cells. After sorting was completed, RNA was collected from sorted cells as described previously and analyzed for the presence of IAβ or HLA-A2.

Statistics
Statistical values were determined using the Student’s t-test (two-tailed, paired) and the Wilcoxon Signed-Rank test. If both tests showed a P value of <0.01 or <0.05 (as indicated), significance was considered highly significant or significant, respectively. Data are presented as mean ± SEM. Differences between treatment groups were determined using the indicated tests with the StatMost Statistical Package (Data XIOM, Los Angeles, CA, USA).


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RESULTS
 
Functional changes of C2D macrophage cells in vivo: cytokine/chemokine profile
Cytokines and chemokines are produced and released by an array of cells and mediate various actions including migration, activation, and development [14 15 16 ]. C2D macrophage cells become more mature when adoptively transferred [9 ]; therefore, we hypothesized that the increased maturity of the cells would affect their cytokine/chemokine profile compared with the cytokine/chemokine profile in vitro. To test this hypothesis, we analyzed the cytokines and chemokines produced by C2D macrophage cells in vivo versus in vitro. CFDA SE-labeled C2D macrophage cells were adoptively transferred (i.p.) to B10, B6, and C2D mice (Table 1) . Three days later, cells were washed from the peritoneal cavity and were sorted on a flow cytometer based on CFDA SE expression (Fig. 1 ). The cells were then plated overnight, and supernatants were analyzed for various cytokines and chemokines. The production of IL-2, IL-4, IL-9, IL-12, IL-13, and IFN-{gamma} by C2D macrophage cells remained below minimum detectable concentrations in vitro and in vivo (data not shown). Low concentrations of IL-1β, GM-CSF, and IL-10 were produced by C2D macrophages stimulated by the in vivo environment (Fig. 2A ). IL-1β was produced by C2D macrophage cells at significantly higher levels after culture in B6 and C2D mice compared with B10 mice (Fig. 2A) . GM-CSF was produced by C2D macrophage cells at significantly higher levels than in vitro only after culture in B6 mice (Fig. 2A) . IL-10 was produced at significantly higher levels by C2D macrophage cells after culture in B6 and C2D mice but not B10 mice (Fig. 2A) .


Figure 1
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  		Figure 1. Parameters used for sorting CFDA SE-labeled C2D macrophage cells after in vivo culture. Three days post-transfer of CFDA SE C2D macrophage cells, peritoneal cavities were washed, cells collected, and CFDA SE-positive cells sorted. To reduce possible contamination by host cells, the lowest 10% of the positive cells was not selected. (A) C2D macrophage cells in vitro (unlabeled control). (B) Representive cytogram for sorting CFDA SE-positive cells (R2; CFDA SE-positive cells collected). (C and D) CFDA SE-labeled C2D macrophage cells were adoptively transferred i.p. into C2D mice. One day post-CFDA SE C2D transfer, mice were injected with B6 or A2T neutrophils containing TRITC-labeled bacteria. Four hours later, peritoneal cavities were washed, and large, granular (Gate R1 in C), double-positive cells (Gate R2 in D) were selectively sorted. FL1, Fluorescence 1.


Figure 2
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  		Figure 2. Cytokines and chemokines secreted by C2D macrophage cells after in vivo or in vitro culture. CFDA SE-labeled C2D macrophage cells were injected i.p. into B10, B6, and C2D mice. Three days post-transfer, peritoneal cavities were washed, cells collected, and CFDA SE-positive cells sorted, and sorted cells were placed in culture overnight. Supernatants were collected, and cytokine/chemokine levels were measured. (A) Cytokines expressed at low levels. (B) Cytokines/chemokines that decreased upon in vivo culture. (C) Cytokines/chemokines expressed at higher levels in vivo than in vitro. minDC, Below minimum detectable concentrations; KC, keratinocyte-derived chemokine. The data are represented as mean ± SEM [n=7; analysis of C2D macrophage cells from seven independent samples (three mice pooled per sample)/mouse strain]. A P value of <0.01 was considered highly significant. *, Treatment group considered different from the other treatment groups covered by the bar.

MCP-1 (CCL2) and RANTES (CCL5), which are expressed during chronic inflammation [17 , 18 ], were produced at high levels by C2D macrophage cells in the in vitro environment but were significantly inhibited with in vivo culture (Fig. 2B) . Similarly, there was a moderate amount of IL-5, an eosinophil differentiation factor, produced by C2D macrophage cells in vitro, which then dropped below detectable levels in vivo (Fig. 2B) .

The production of MIP-1{alpha} (CCL3), TNF-{alpha}, and IL-6 by C2D macrophage cells was significantly increased upon in vivo culture (Fig. 2C) . The largest increase was seen in MIP-1{alpha} production, possibly as a result of the role of MIP-1{alpha} in the induction of IL-1β, IL-6, and TNF-{alpha} [19 ]. Although the production of KC (CXCL1), a neutrophil chemoattractant [20 21 22 23 ], significantly increased upon adoptive transfer to B6 or C2D mice, the in vivo environment of B10 mice caused a significant decrease in the production of KC by C2D macrophage cells (Fig. 2C) . The levels of IL-6, TNF-{alpha}, and KC were also elevated at the transcriptional level as measured by RT-PCR (data not shown).

Functional changes of C2D macrophage cells in vivo: phagocytosis
As C2D macrophage cells altered their cytokine secretion after adoptive transfer, we hypothesized that the in vivo environment would enable C2D macrophage cells to become better phagocytes. To assess phagocytic activity, we analyzed two mechanisms of phagocytosis: direct phagocytosis of small, free particles using fluorescent beads and macrophage phagocytosis of PMNs, characteristic of macrophage phagocytosis during inflammation. C2D macrophage cells were significantly more phagocytic in vivo than cells in vitro (Fig. 3A ). We assessed phagocytosis up to 3 days post-transfer of C2D macrophage cells and determined that 1 day in vivo was sufficient for C2D macrophage cells to respond to their environment. Direct and inflammation-like phagocytosis was enhanced (Fig. 3B and 3C) . Additionally, assessment of cytocentrifuge preparation of cells collected from the peritoneal cavity revealed numerous macrophages that had phagocytosed neutrophils or cellular debris (Fig. 4 ).


Figure 3
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  		Figure 3. Phagocytosis by C2D macrophage cells in vivo and in vitro. In vivo: CFDA SE-labeled C2D macrophage cells were adoptively transferred i.p. into B6 mice. One, 2, or 3 days post-transfer of CFDA SE C2D macrophage cells, mice were injected with fluorescent beads or TRITC-labeled B6 neutrophils as described in Materials and Methods. Four hours later, peritoneal cavities were washed, and phagocytosis was measured by flow cytometry. In vitro: Fluorescent beads or TRITC-labeled B6 neutrophils were added to C2D macrophage cells and incubated at 37°C (experimental) or 4°C (control) for 4 h. Phagocytosis was measured by flow cytometry. (A) Phagocytosis on all days combined. (B) Phagocytosis of beads by C2D macrophage cells. (C) Phagocytosis of neutrophils by C2D macrophage cells. The data are represented as mean ± SEM (n=4 mice per time-point in vivo, analysis of C2D macrophage cells from four different mice; n=5 in vitro, analysis of five independently treated plates of C2D macrophage cells). *, A significant difference between phagocytosis in vivo than in vitro. A P value of <0.05 was considered significant.


Figure 4
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  		Figure 4. Neutrophil phagocytosis by macrophages in vivo. One day post-CFDA SE C2D macrophage cell transfer, mice were injected with B6 neutrophils containing TRITC-labeled bacteria. Four hours later, peritoneal cavities were washed, and slides were made immediately from the unsorted peritoneal washes to identify neutrophils phagocytosed by macrophages. Slides were stained using Wright’s stain. Six random fields are shown (n=65 fields). Top two fields and middle left: x264 original magnification. Middle right and bottom two fields: x660 original magnification. Arrows point to phagocytosed cells or debris.

After in vivo culture in B10, B6, or C2D mice, RNA from sorted C2D macrophage cells (IAβ–/–) was analyzed for the transcription of IAβ to confirm the absence of IAβ in our sorted C2D macrophage cells. However, to our surprise, the presence of IAβ was detected in cells sorted from B10 and B6 mice, both of which are IAβ+/+. We used conservative parameters to sort CFDA SE-positive C2D macrophage cells (Fig. 1) . CFDA SE reacts with amine groups on proteins and forms stable, covalent bonds to make the target cells fluorescent [24 , 25 ]. This technique is preferable to the use of lipophilic dyes, as the covalent protein bonds do not allow for transfer of the fluorochrome as can occur with lipophilic dyes [26 , 27 ]. Therefore, we were confident that the C2D macrophage cells were not contaminated by recipient cells. As a result of the increased phagocytic ability of C2D macrophage cells in vivo, we hypothesized that IAβ was detectable in C2D macrophage cells, as they phagocytosed recipient host cells after adoptive transfer. To test this hypothesis, we analyzed C2D macrophage cells for the presence of IAβ and HLA-A2 mRNA after exposure to neutrophils that came from HLA-A2+ or IAβ+ animals. HLA-A2 is a human MHCI molecule absent in C2D mice as well as C2D macrophage cells but a transgene expressed in A2T mice (Table 1) [10 ]. TRITC fluorescent neutrophils were collected from B6 mice (IAβ-positive) or A2T mice (HLA-A2-positive). These neutrophils were then injected separately into the peritoneal cavity of C2D mice already containing CFDA SE-labeled C2D macrophage cells, which were collected 4 h later and positively sorted based on size as well as CFDA SE and TRITC expression. RNA from the sorted, double-positive cells was then analyzed, and the presence of IAβ or HLA-A2 was confirmed by RT-PCR (Fig. 5 ).


Figure 5
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  		Figure 5. Detection of neutrophil RNA in C2D macrophage cells in vivo. CFDA SE-labeled C2D macrophage cells were adoptively transferred i.p. into C2D mice. One day post-CFDA SE C2D transfer, mice were injected with B6 or A2T neutrophils containing TRITC-labeled bacteria. Four hours later, peritoneal cavities were washed, and double-positive cells were selectively sorted as described in Materials and Methods and Figure 1 . The presence of IAβ or HLA-A2 was confirmed by RT-PCR and gel electrophoresis. Normalized densitometric values were determined by cytokine product/β-actin product, which were electrophoresed on the same gel and assessed at the same time. Images of β-actin products were cut and pasted to facilitate viewing. (A) Detection of neutrophil HLA-A2 mRNA. (B) Detection of neutrophil IAβ mRNA. The data presented are representative of two independent experiments per neutrophil type with all four experiments showing similar outcomes.


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DISCUSSION
 
The responsiveness of C2D macrophage cells in vivo demonstrates the power of the in vivo environment to regulate cells in ways not possible in vitro and is consistent with the hypothesis that microenvironments regulate macrophages [28 29 30 31 32 ]. C2D macrophage cells do not become functional or differentiate in vitro even when stimulated with M-CSF or G-CSF and IL-4 at concentrations that induce primary bone marrow cells to differentiate [9 ]. Therefore, other stimuli, such as exposure to tissue or extracellular matrix (ECM) molecules, are necessary to induce the C2D macrophage cells to differentiate and function. In support of this hypothesis, Mukaida et al. [33 ] demonstrated that the interaction between leukocytes and endothelium through β2-integrin adhesion molecules was important for maximal TNF-{alpha} production through the NF-{kappa}B pathway. In addition, adherence of human monocytes to various ECM proteins, some of which are found on endothelium, leads to the expression of several macrophage genes [34 , 35 ]. As C2D macrophage cells were originally differentiated in vitro, their ability to transcribe significant amounts of TNF-{alpha} and possibly other cytokines in vitro may not have yet been properly developed as a result of lack of contact with the endothelium. Sorted C2D macrophage cells directly isolated from the peritoneum, where the cells would have been exposed to many tissue molecules, display an altered cytokine/chemokine profile as compared with cells maintained and even labeled with CFDA SE in vitro. Moreover, these cells are also distinguishable from normal peritoneal macrophages, which do not express activated macrophage markers [36 ] and produce low cytokine levels [37 ]. Although these data are consistent with this hypothesis, studies with integrin-deficient mice will be necessary to clearly define the role of integrins during cytokine up-regulation.

IL-1β, GM-CSF, and IL-10 were expressed at low levels by C2D macrophage cells after in vivo culture compared with C2D macrophage cells cultured in vitro (Fig. 2A) . Moreover, IL-1β and IL-10 were secreted at higher concentrations by C2D macrophage cells after culture in B6 and C2D mice, compared with B10 mice. These data are consistent with observations that TLR4 influences the production of IL-1β [38 ] and GM-CSF [39 ] and suggest that tissue expression of TLR4 also impacts macrophage function, not just TLR4 expression by the macrophages themselves.

Although we saw an up-regulation of some cytokines in C2D macrophage cells after adoptive transfer, some cytokines were actually down-regulated. In fact, some of the cytokines expressed by C2D macrophage cells in vitro but not in vivo are classified as Th2 cytokines [40 , 41 ] (Fig. 2B) . MCP-1 (CCL2) and RANTES (CCL5) are chemokines that bind CCR1 and CCR5 and can act as chemoattractant for monocytes and neutrophils [14 , 42 ]. RANTES is also a chemoattractant for basophils and eosinophils [14 ]. The differential expression of these β chemokines by C2D macrophage cells may correlate with the altered phenotype of these cells within the in vivo environment. MCP-1 and RANTES have been reported to be synthesized by numerous fibroblasts found throughout the body [18 ]; thus, the immature phenotype of C2D macrophage cells in vitro (based on their low-level expression of mature macrophage markers F4/80 and CD11b and mid-expression level of Mac-2) [9 ] may correlate with the high expression level of MCP-1 and RANTES in vitro, followed by a decrease in expression of these chemokines upon maturation in vivo (Fig. 2B) . An alternative explanation may be that MCP-1 is associated with Th2, and MIP-1{alpha} is associated with a Th1 response [15 , 43 ], and upon in vivo culture, Th1 cytokines are enhanced significantly, and Th2 cytokines are expressed at only low-to-moderate levels. This hypothesis is supported by the fact that the lung is often considered an M2 (Th2) environment [44 ]. Moreover, CCL2 is transcribed earlier in the lungs of B10 mice than CCL3 after adoptive transfer of C2D macrophage cells and challenge with gram-negative bacteria [45 ]. In the same manner, IL-5, an eosinophil-differentiation factor [40 ], is significantly down-regulated upon in vivo culture (Fig. 2B) . The initial production of IL-5 may be a result of the immature phenotype of C2D macrophage cells, which differentiate in vivo and cease to produce IL-5.

MIP-1{alpha}, TNF-{alpha}, and IL-6 levels were increased to high levels upon in vivo culture of C2D macrophage cells (Fig. 2C) and can be classified as proinflammatory [19 ]. They are involved in mediating inflammation as well as regulatory immune functions [19 , 41 , 42 ]. IL-6 was produced by C2D macrophage cells at a significantly higher level after adoptive transfer to B6 mice than to B10 mice (Fig. 2C) . This is consistent with the tissue impact on macrophage IL-1β and GM-CSF secretion (above) as well as with studies showing that lower TLR4 levels correlate with lower IL-6 levels [46 , 47 ]. The production of KC by C2D macrophage cells was also influenced by the mouse strain in a TLR4-dependent manner (Fig. 2C) . The murine gene KC contains two NF-{kappa}B motifs, which allow for induction by LPS, but no IFN regulatory sequences have been identified [20 ]. Thus, LPS is able to induce KC production in wild-type mice; however, it is not able to induce KC in TLR4 mutant mice [48 ]. The down-regulation of KC in B10 mice appears to be consistent; however, C2D macrophages contain functional TLR4 molecules and are constitutive producers of KC in vitro, which suggest that the production of KC by C2D macrophage cells is dependent on interactions with the host.

In our studies, only 1 day of in vivo culture was necessary to induce changes in phagocytosis in B6 mice. It is possible that receptors such as selectins and integrins expressed in the peritoneum signaled cytoskeletal changes [49 ]. For example, CD11b expression was increased significantly on C2D macrophage cells in vivo [9 ], which could enhance phagocytosis by binding to cleaved C3 or ICAM [49 ]. Increased secretion of IL-1 and TNF-{alpha} (Fig. 1) could also enhance phagocytosis [32 ]. It is possible that phagocytosis by C2D macrophages would have been different in B10 or C2D mice. However, as we observed few mouse strain-dependent changes in phenotype expression in C2D macrophage cells after adoptive transfer, we did not pursue those studies at this time.

The adoptively transferred C2D macrophage cells phagocytosed recipient cells after adoptive transfer. We detected mRNA unique to the recipient in reisolated C2D macrophage cells 4 h after they were isolated (Fig. 5) . Although we saw evidence of phagocytosis, we saw no intact neutrophils inside C2D macrophage cells (Fig. 4) . Therefore, our observations are consistent with suggestions that these cells are degraded rapidly [50 ]. In spite of the rapid cellular degradation, our data suggest that RNA from the phagocytosed cells may be more stable than assumed, and the implications of these data have yet to be explored.

In summary, the peritoneal microenvironment alters the capabilities of C2D macrophage cells, causing them to express primarily a functional, proinflammatory phenotype. Thus, these data support the hypothesis that macrophage heterogeneity is determined by the local microenvironment, and their potential use in therapy may be tissue/site-specific.


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ACKNOWLEDGEMENTS
 
This project has been supported by NIH grants AI55052, AI052206, RR16475, RR17686, and NASA NAG2-1274, the Kansas Agriculture Experiment Station, and the Terry C. Johnson Center for Basic Cancer Research. This is Kansas Agriculture Experiment Station Publication 07-298-J. We thank Mrs. Tammy Koopman for her assistance with FACS analysis and cell sorting. We also thank Ms. Whitney Mordica and Mrs. Alison Fedrow for their laboratory assistance with these studies and Dr. Mark Haub’s laboratory for their help using the Luminex equipment.

Received June 8, 2007; revised September 21, 2007; accepted November 1, 2007.


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