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Colleges of
*
Pharmacy and
Veterinary Medicine, Chungbuk National University, Cheongju, South Korea;
Department of Pharmacy, Sahm-Yook University, Seoul, South Korea; and
Laboratory of Molecular Immunoregulation, National Cancer Institutes, Frederick, MD 21702
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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and -
genes
and to be committed to the T cell lineage. Thymic DCs play a key role in presenting self Ags to developing thymocytes. The preceding observations have suggested that thymic DCs, rather than deriving from myeloid precursors, actually develop from the same thymic progenitors, pro-T1 and -T2 cells, as do T lymphocytes (16).
Thymic macrophages play an important role in eliminating apoptotic thymocytes (17), which die at a high rate because of failure of their Ag receptors to recognize self Ags with appropriate avidity. Part of the recognition of apoptotic thymocytes by thymic macrophages is via scavenger receptors (18). Thymic macrophages are heterogeneous in surface markers (19). The origin of thymic macrophages has not been established. On one hand, they could derive from blood monocytes; however, it is also possible that they develop locally based on evidence of myelopoiesis in the thymus (20). If thymic macrophages were generated locally, it would be expected that they derive from multipotential HSCs rather than from pro-T cells, because the latter failed to generate macrophages under the conditions tested previously (reviewed in Ref. 16).
Here, we report that early T progenitors have the potential to generate macrophages in in vitro culture incorporating M-CSF plus IL-6 plus IL-7. Pro-T1 and -T2 cells were shown, through cell sorting and single-cell cloning, to generate macrophages under these conditions, whereas pro-T3 and pro-T4 cells had lost this capacity. Our results extend the lineage potential of early T progenitors and suggest that thymic macrophages, like thymic DCs, could be generated within the thymus from a common progenitor.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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The mouse thymic stromal cell line, TFGD, was obtained from a thymoma mass that spontaneously developed in a p53-/- mouse. Briefly, a single-cell suspension of the tumor was prepared by treatment with trypsin and cultured in RPMI 1640 supplemented with 2 mM glutamine, 100 U/ml penicillin, 100 µg/ml streptomycin, and 10% heat-inactivated FBS (HyClone, Logan, UT) in a tissue culture plate. Vigorously growing adherent cells were subcultured three times a week thereafter. At the 28th subculture, this cell line was identified to produce cytokines that induce differentiation of thymic progenitor cells into macrophages.
Cytokines and mAbs
Recombinant human M-CSF, mouse GM-CSF, mouse IFN-, mouse
IL-6, and mouse IL-7 were purchased from PeproTech (Rocky Hill, NJ).
The mAbs recognizing murine cell surface markers, anti-CD11b (clone
M1/70), anti-CD11c (clone HL3), anti-CD25 (clone 7D4),
anti-CD40 (clone 3/23), anti-CD44 (clone IM7), anti-CD45
(clone 30F11.1), anti-B220 (clone RA3-6B2), anti-HSA (clone
M1/69), anti-ICAM-1 (clone 3E2),
anti-I-Ab (clone AF6-120.1), anti-B7-1
(clone 16-10A1), anti-B7-2 (clone GL1), and anti-Gr-1 (clone
RB6-8C5) were purchased from BD PharMingen (San Diego, CA). Anti-F4/80
(clone A3-1) was purchased from Serotech (Oxford, U.K.). Anti-Dec-205
and anti-CSF1R were provided by Drs. K. Komschlies and J. Keller
(National Cancer Institutes, Frederick, MD), respectively. As
isotype-matched control Abs, anti-TCR-V4 (clone KT4),
anti-TCR-V2 (clone B20.6), anti-TCR-V14 (clone 14-2),
anti-TCR-V3 (clone KJ25), anti-TCR-8.1&2 (clone MR5-2)
and anti-TNP (clone G235-1) were purchased from BD PharMingen and
were used respectively.
Preparation and sorting of fetal thymocytes
C57BL/6 mice were mated overnight, and plugs were checked the following day, which was designated as day 1 of gestation. At day 14 or 15 of gestation, mothers were sacrificed by CO2 asphyxiation and embryos by chilling on ice. Fetal thymus lobes were removed under dissecting microscope. Fetal thymocytes were prepared from fetal thymus lobes by gentle disruption after treatment with 0.2% collagenase (Sigma, St. Louis, MO) for 1 h at 37°C as previously described (12). In some experiments, fetal thymocytes were sorted into four populations (pro-T1 to pro-T4) on the basis of CD44 and CD25 expression on a FACStarPlus or FACSCalibur (BD Biosciences, Mountain View, CA) as previously described (12). The purity of sorted populations was generally >98%.
Generation of macrophages from fetal thymocytes
Adherent cells were first removed from total day-14 fetal thymocytes by plastic adherence after incubating fetal thymocytes overnight in a 6-well or 24-well plate in a culture medium supplemented with the culture supernatant of TFGD (50%, final concentration) or with defined amounts of cytokine(s). After the adherent cell-depleted thymocytes were counted, we adjusted the cell number (1 or 2 x 105/ml) with the same medium and then distributed them to wells of a 24-well tissue culture plate (1 ml/well). Cells were fed with the same medium every 3 to 4 days by replacing half of the culture medium with fresh medium. For sorted populations of fetal thymocytes, cells were cultured directly in a 24-well tissue culture plate (1 x 105/well) in a culture medium supplemented with the culture supernatant of TFGD.
Phenotypic analysis
Macrophages (2 x 105) were washed in a staining solution of PBS containing 5% FBS and 0.1% NaN3 and then resuspended in 50 µl of staining solution containing 1.0 µg of anti-CD16/CD32 mAbs (clone 2.4G2) and 10% normal mouse serum to block nonspecific binding of Abs to Fc receptors. Cells were incubated for 10 min on ice, mixed with 50 µl of prediluted Ab solution, and then incubated for another 20 min on ice. Unbound Abs were removed by washing the cells twice with staining solution. For biotinylated Abs, the cell pellet was resuspended in 50 µl of PE- or FITC-conjugated avidin solution, incubated for 10 min on ice, and then washed twice with staining solution. For unconjugated Abs, Dec-205 and CSF1R, the cell pellet was resuspended in 50 µl of FITC-goat anti-rat IgG (BD PharMingen), incubated for 10 min on ice, and then washed twice with staining buffer. After a final washing, cells were fixed in 1% paraformaldehyde in PBS, and flow cytometric analysis was performed on a FACSCalibur (BD Biosciences). Dead cells were gated out by their low forward-angle light scatter intensity. In most analyses, 10,000 cells were scored.
Phagocytic activity
Macrophages were cultured overnight in slide chambers (0.5–1 x 106/chamber) in a volume of 1.0 ml and were then mixed with 100 µl/well of IgG-opsonized sheep RBC (SRBC; 5%). The slide chambers were incubated for 1 h at 37°C, rinsed with PBS, and then treated with ACK lysis buffer for 3 min to lyse uningested SRBC. Opsonized SRBC were prepared by incubating SRBC with a 1:256-diluted mouse anti-SRBC IgG Ab (Cordis, Miami, FL) for 30 min at 37°C in a shaking water bath.
NO production
Macrophages were cultured with LPS (Difco, Detroit, MI; 10
ng/ml), IFN- (100 U/ml), or both in a 96-well flat-bottom microtiter
plate (2 x 105 cells/well) in a total
volume of 200 µl. After overnight stimulation, 50 µl of cell-free
supernatant was incubated with 50 µl of Griess reagent (1%
sulfanilamide/0.1% naphthylenediamine dihydrochloride/2.5%
H3PO4) at room temperature
for 5 min, and the absorbance at 550 nm was determined in a Dynatech
MR500 microplate reader (Dynatech Laboratories, Chantilly, VA). The
concentration of NO2- was
determined from a least squares linear-regression analysis of a sodium
nitrite standard curve.
APC function
APC function of macrophages derived from pro-T cells was assessed by examining their ability to stimulate proliferation of T cells with anti-CD3. T cells were isolated from the spleen of C57BL/6 mice by depleting adherent cells with nylon wool adherence and then treating the adherent cell-depleted cells with anti-Ia mAb (clone 25-9-3s; American Type Culture Collection, Manassas, VA) plus rabbit serum (Low-Tox-M; Cedarlane Laboroatories, Ontario, Canada). Purified T cells (1 x 106/ml) were mixed with anti-CD3 mAb (50 ng/ml; BD PharMingen), and then 100 µl of the cell suspension was added to each well of U-bottom plates containing pro-T derived macrophages. Adherent cells that were isolated from the spleen cells of C57BL/6 mice by plastic adherence (37°C, 2 h) were used as a control APC. DNA synthesis was measured by [3H]thymidine (DuPont Pharmaceuticals, Wilmington, DE) incorporation (1 µCi/well) for the final 6 h of the 3-day culture period.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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A thymic stromal cell line, TFGD, was derived from a thymic tumor mass that developed spontaneously in a p53-/- mouse. At the 28th passage, supernatant from the TFGD line was observed to induce pro-T cells to differentiate into a cell type that did not resemble a lymphoid cell, and which was later identified as a macrophage. As a source of early T progenitors, we used day-14 embryonic fetal thymocytes, which are composed of the first two stages of pro-T cells, pro-T1 and pro-T2, in approximately equal proportions. To insure that the macrophages generated in this culture system did not derive from macrophages in the starting population, adherent cells in the fetal thymocyte preparations were first removed by plastic adherence after being cultured overnight in a culture medium containing the culture supernatant from TFGD cells (50%, final concentration). The adherent cell-depleted fetal thymocytes were then cultured in the same type of medium for additional 7–8 days. Most of the fetal thymocytes formed a layer of adherent cells when observed at day 4 from the initiation of the culture, and the adherent cells, appearing to undergo cell division, differentiated into macrophages. Approximately 6 x 105 macrophages could be recovered consistently at day 8 from 2 x 105 adherent cell-depleted day-14 fetal thymocytes.
Shown in Fig. 1
are the phenotypic and
functional characteristics of the macrophages generated after 8 days of
culture of pro-T cells in supernatant from TFGD cells. Pro-T-derived
macrophages exhibited variable degrees of cytoplasmic vacuolation but
lacked cytoplasmic granules, as shown in forward and side scatter
profiles. As shown in Fig. 1A, the cells expressed surface
markers typical of mouse macrophages including CD11b (Mac-1), receptors
for CSF-1 (CSF-1R), CD24 (heat-stable Ag), CD44 (pgp-1), CD45
(leukocyte common Ag), and CD54 (ICAM-1). Pro-T-derived macrophages did
not express lymphoid lineage markers such as CD25 and CD45R (B220). The
granulocyte lineage marker, Gr-1, was not expressed. DC-restricted
markers, Dec-205 and CD11c, were also negative in the macrophages,
although a low level of expression was observed for F4/80, which is
also expressed on some macrophages. We examined surface molecules
important in interacting with T cells and observed high levels of
expression of both the costimulatory molecule B7-1 (CD80) as well as
the activating receptor CD40. B7-2 (CD86), another costimulatory
molecule, was not expressed, and the constitutive level of MHC class II
(I-Ab) was low. However, the level of MHC class
II was markedly up-regulated upon stimulation with IFN-, as shown in
Fig. 1B.
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and LPS stimulation (Fig. 1
C). The macrophages produced higher amounts of NO than the
control mouse macrophage cell line ANA-1, which was established by
infecting fresh bone marrow-derived cells from C57BL/6 with the J2
(v-raf/v-myc) recombinant retrovirus (21) and
has been well characterized to produce NO. Pro-T-derived macrophages
were also highly phagocytic for IgG-opsonized SRBC, as shown in Fig. 1D. APC function of pro-T-derived macrophages was weak, as
shown in Fig. 2, adding to the criteria
distinguishing these macrophages from DCs, which are potent
APCs.
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Mouse embryonic thymocytes were sorted into four populations,
pro-T1 to pro-T4, the first four stages of development, and then
examined for their developmental potential to macrophages. Pro-T1
cells, cells in the the most primitive stage, effectively generated
macrophages when cultured in a medium with TFGD culture supernatant
(Fig. 3, upper panel). Even
pro-T2 cells could generate macrophages (Fig. 3, lower
panel) under these conditions, although fewer, when compared with
pro-T1 cells, initiated with the same cell number as can be seen
comparing the results found in Fig. 3, upper vs lower
panels. Generation of macrophages from pro-T1 and T2 cells was
further confirmed by a single-cell cloning assay we recently used to
determine the clonal origins of NK, 
, and 
T cells
(12). Pro-T1 and T2 cells were sorted, plated at a
frequency of one cell per well in a round-bottom microtiter plate, and
then cultured for an additional 7 days in a medium supplemented with
TFGD culture supernatant. Microscopic observation of the wells showed a
range of 48–74% (two experiments) of pro-T1 cells vs 6–15% (three
experiments) of pro-T2 cells differentiated into adherent cells
with morphology of macrophages (data not shown). The later stages
(pro-T3 and pro-T4) were unable to generate macrophages under these
conditions (data not shown). Single cells underwent very few cell
divisions during this culture period, just one to three divisions for
pro-T1 and none or one division for pro-T2 cells. To verify that single
cells generated macrophages, we observed that the cells readily
phagocytosed fluoresced, opsonized latex beads.
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To identify the cytokines responsible for the generation of
macrophages from early T progenitors, we first assayed for a number of
cytokines in the TFGD culture supernatant by ELISA. High levels of
M-CSF and IL-6 were detected, whereas no GM-CSF, IL-4, IL-5, IL-9, or
IL-13 was detectable (data not shown). Because M-CSF was present, and
it is an effective inducer of macrophage generation from bone marrow
cultures and IL-6 is an effective cofactor in macrophage generation, we
examined the ability of M-CSF and IL-6 alone and in combination with
other cytokines to induce the development of macrophages from pro-T
cells. As shown in Fig. 4, M-CSF alone or
IL-6 alone failed to induce differentiation of day-14 thymocytes into
macrophages. Combining IL-6 with M-CSF could induce macrophage
differentiation from early T progenitors, although the
differentiation-inducing activity was low compared with that of TFGD
culture supernatant. Combining IL-7 with M-CSF and IL-6 resembled TFGD
culture supernatant in strongly increasing the differentiation to
macrophages. Comparison of TFGD culture supernatant with the mixture of
IL-7, M-CSF, and IL-6 showed that the defined cytokine mixture induced
about half the frequency of macrophage potential from individual pro-T
cells, suggesting that TFGD culture supernatant may contain additional
activities.
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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stimulation. The macrophages expressed a low level
of class II MHC molecules that increased significantly following
IFN- stimulation. We show that macrophages could be generated from sorted populations of pro-T1 or T2 cells. Limiting dilution analysis indicated that sorted pro-T1 and T2 cells showed a range of cloning efficiency of 48–74% and 6–15%, respectively, making it unlikely that the macrophages were generated from contaminating multipotential stem cells. An earlier study showed that mouse fetal thymocytes could generate macrophages in vitro when cultured with a stromal cell line (5) and was interpreted to indicate the presence of multipotential stem cells that have been shown to be present in the fetal thymus (4), whereas our study defines conditions under which true pro-T cells can generate macrophages.
Our results extend the lineage potential of early T progenitors in the thymus. As reviewed by Shortman et al. in detail (16), the c-kit+CD44+CD25- (pro-T1) cells have previously been shown to have the capacity to form B cells, NK cells, and thymic DCs, as well as T cells, but not erythroid or myeloid cells, under the conditions tested at that time (15). The next downstream precursor, pro-T2 cells, appeared to have lost B cell and NK cell potential but still retained the capacity to form DCs. Thus, thymic DCs are now thought to derive from the common lymphoid precursor rather than from a myeloid precursor. Our results show that, like DCs, which were previously thought to be of myeloid origin, macrophages, too, can derive from a "lymphoid" progenitor.
Some parallels between B and T cell development can now be seen with regard to the potential to generate other cell types. A bipotential B macrophage progenitor has been demonstrated previously (22). Those bipotential B macrophage progenitors, like the T macrophage progenitors in this study, respond to IL-7 (23). DCs have also been shown to be generated from pro-B cells (24) under similar conditions required to generate DCs from pro-T cells. In both T and B lineages, VDJ recombination marks a stage of loss of macrophage and DC potential. If the onset of VDJ recombination was coupled to lymphoid commitment, it could explain why, in our study, a lower proportion of pro-T2 cells had macrophage potential than did pro-T1 cells, because VDJ recombination accelerates at the pro-T2 stage (25).
M-CSF alone was unable to induce macrophage differentiation from early T progenitors, whereas it alone is sufficient to induce bone marrow precursors to generate macrophages. The combination of M-CSF and IL-6 induced significant macrophage differentiation from pro-T cells, a phenomenon similar to the observation of Jansen et al. that IL-6 is required for optimal monocytic colony formation by bone marrow cells (26). Addition of IL-7 to M-CSF plus IL-6 dramatically increased the frequency of macrophages generated from pro-T cells and presumably reflects the dependency of pro-T cells on survival signals from IL-7 (27, 28). The IL-7 effect also distinguishes pro-T cells from bone marrow cells in the signals required for macrophage generation and may explain the previous failures to detect macrophage generation from this cell type. We noted that the TFGD culture supernatant was about twice as efficient in inducing macrophage differentiation from pro-T cells, suggesting it may contain activities in addition to IL-7, M-CSF, and IL-6. While the current manuscript was under review, a study (29) was published showing that introducing a transgenic IL-2 receptor into lymphoid progenitors from bone marrow enabled them to be diverted into the myeloid lineage in a cytokine mixture. Our study shows that macrophage diversion can be induced through receptors naturally expressed on pro-T cells.
It remains to be shown whether in vivo thymic macrophages, like thymic DCs, derive from pro-T cells. However, this possibility is supported by findings that the three cytokines that we show generate macrophages in vitro are produced in the thymus: M-CSF (30), IL-6 (31), and IL-7 (32). Local production of macrophages would be a means of coordinating the numbers of thymocytes with the numbers of macrophages required to dispose of dead thymocytes, which some estimate as high as a third of the thymocytes dying each day.
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Acknowledgments |
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Footnotes |
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2 Address correspondence and reprint request to Dr. Chong-Kil Lee, College of Pharmacy, Chungbuk National University, Cheongju 361-763, South Korea.
3 Abbreviations used in this paper: HSC, hemopoietic stem cell; DC, dendritic cell.
Received for publication April 25, 2000. Accepted for publication March 2, 2001.
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References |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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RII/III contains precursors of T lymphocytes and natural killer cells. Cell 69:139.[Medline]
- T cell potential. Cell. Immunol. 191:139.
gene rearrangement in interleukin-7 receptor knock out mice. Immunol. Lett. 57:9.[Medline]
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)
and a control macrophage cell line ANA-1 cells (
) were stimulated
overnight with LPS (10 ng/ml), IFN-
