Differential rates of replacement of human dermal dendritic cells and macrophages during hematopoietic stem cell transplantation - PubMed (original) (raw)

Comparative Study

. 2009 Feb 16;206(2):371-85.

doi: 10.1084/jem.20081633. Epub 2009 Jan 26.

Florent Ginhoux, Xiao-Nong Wang, Venetia Bigley, Michal Abel, Ian Dimmick, Sarah Bullock, Marcos Grisotto, Trevor Booth, Peter Taub, Catharien Hilkens, Miriam Merad, Matthew Collin

Affiliations

Comparative Study

Differential rates of replacement of human dermal dendritic cells and macrophages during hematopoietic stem cell transplantation

Muzlifah Haniffa et al. J Exp Med. 2009.

Abstract

Animal models of hematopoietic stem cell transplantation have been used to analyze the turnover of bone marrow-derived cells and to demonstrate the critical role of recipient antigen-presenting cells (APC) in graft versus host disease (GVHD). In humans, the phenotype and lineage relationships of myeloid-derived tissue APC remain incompletely understood. It has also been proposed that the risk of acute GVHD, which extends over many months, is related to the protracted survival of certain recipient APC. Human dermis contains three principal subsets of CD45(+)HLA-DR(+) cells: CD1a(+)CD14(-) DC, CD1a(-)CD14(+) DC, and CD1a(-)CD14(+)FXIIIa(+) macrophages. In vitro, each subset has characteristic properties. After transplantation, both CD1a(+) and CD14(+) DC are rapidly depleted and replaced by donor cells, but recipient macrophages can be found in GVHD lesions and may persist for many months. Macrophages isolated from normal dermis secrete proinflammatory cytokines. Although they stimulate little proliferation of naive or memory CD4(+) T cells, macrophages induce cytokine expression in memory CD4(+) T cells and activation and proliferation of CD8(+) T cells. These observations suggest that dermal macrophages and DC are from distinct lineages and that persistent recipient macrophages, although unlikely to initiate alloreactivity, may contribute to GVHD by sustaining the responses of previously activated T cells.

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Figures

Figure 1.

Figure 1.

Characterization of resident dermal APC. (A) Strategy used in the characterization of resident CD45+HLA-DR+ dermal cells showing successive gating on DAPI-live cells, CD45+ cells, and HLA-DR+ cells. Progressive refinement of SSCloAFlo and SSChiAFhi fractions is depicted at each step by the corresponding panel below. CD45+ cells include macrophages, DC, mast cells, and lymphocytes. Mast cells (high SSC) are HLA-DR−, as are the majority of lymphocytes (low SSC), and are easily excluded by the final HLA-DR gate. AF was recorded in the FL1 channel (488-nm laser and 530/30 band-pass filter). Similar results were found in >50 independent preparations of skin. (B) Further analysis of SSCloAFlo and SSChiAFhi fractions of CD45+HLA-DR+ cells by expression of CD14+ and CD1a+. Blue indicates the position of isotype controls and red the expression of CD14/CD1a by each fraction. Three principal populations were sorted and show distinct morphological appearances by Giemsa staining (right). Bar, 20 µm. A representative example of three experiments is shown. (C) Surface phenotype of resident CD45+HLA-DR+ dermal cells. Gates were placed as described in A and B using CD45, HLA-DR, SSC, AF CD1a, and CD14 to define macrophages (mac), CD14+ DC, and CD1a+ DC. CD14 expression on DC cells was analyzed by gating on CD1a+ and CD1a− cells. All other markers for CD1a+ DC and CD14+ DC were determined by gating on CD14− and CD14+ populations, respectively. Staining was performed at least six times on different skin preparations with similar results. (D) Identification of large melanosome-laden macrophages by CD45, HLA-DR, and FXIIIa. Freshly isolated APC prepared on cytospin were simultaneously stained with antibodies to CD45 (green), HLA-DR (red), and FXIIIa (blue). After immunofluoresence imaging the coverslip was removed and the slide stained with Giemsa. FXIIIa staining is restricted to macrophages that are a subset of the HLA-DR+ APC in the preparation (red). A number of small agranular HLA-DR+ DC with reniform nuclei are FXIIIa negative. All HLA-DR+ cells and a number of small lymphocytes in the field are highlighted by CD45 staining. Bar, 20 µm. This finding was reproduced three times.

Figure 2.

Figure 2.

Functional properties of dermal myeloid cells. (A) Differential migration of dermal myeloid cells. 1-cm square dermal sheets were freshly digested with collagenase or incubated in RPMI 10% FCS for 72 h in low adhesive culture plates, after which migrant cells were collected and the remnant dermis was digested to release nonmigrating cells. CD45+HLA-DR+ gated cells were analyzed by SSC and AF and further separated into CD14+/− fractions as described in Fig. 1. Populations a–e were sorted by flow cytometry for morphological analysis as shown. CCR7 expression was determined on freshly isolated populations. The experiment was repeated 12 times with similar findings. Bar, 20 µm. (B) Phagocytosis of FITC dextran by dermal myeloid cells. 5 × 105 collagenase-digested dermal cells were incubated with 1 mg/ml of FITC-coated dextran particles at 37 or 4°C for 1 h in RPMI containing 10% FCS. Cells were harvested and washed before immunostaining. Fluorescence histograms show a representative example. For the bar charts, all three are independent experiments. ΔMFI, change in geometric mean fluorescence compared with no dextran control. (C) Adherence of dermal myeloid cells. 5 × 105 collagenase-digested cell suspensions were cultured overnight in RPMI containing 10% FCS. Nonadherent cells and washes were collected and pooled. Adherent cells were removed by trypsinization. Flow cytometric analysis was performed using SSC, AF, CD14, and CD1a according to Fig. 1. A representative example is shown with the cumulative results of five independent experiments. Bars represent the means.

Figure 3.

Figure 3.

Effect of conditioning therapy on recipient dermal DC and macrophages. (A) Representative flow cytometry analysis of dermal cell suspensions derived from clinical biopsies by collagenase digestion before and after conditioning. Selective depletion of SSCloAFloCD14−CD1a+ DC is evident. (B) Paired analysis of dermal cells obtained before and after conditioning from a total of 27 patients showing a decline in CD1a+ DC but preservation of CD14+ DC and macrophages. Enumeration of cells per unit area was achieved using a standard 2-mm punch of shaved dermis freshly digested into single cell suspension with collagenase. The total number of cells present in each fraction was quantified with the aid of Trucount fluorescent beads. (C) Subgroup analysis of 17 patients treated with reduced intensity and 10 patients with full intensity conditioning showing similar depletion of dermal DC in both groups. P <0.001 for before and after counts (Wilcoxon rank sum test). There is also a slight but nonsignificant increase in CD14+ DC with full intensity conditioning. Error bars indicate SD. (D) Immunofluorescence images of skin before and after conditioning showing maintenance of CD163+ perivascular macrophages. AF in the green channel can be seen in some cells (arrows). Bar, 50 µm. Two representative examples of six patients are shown. (E) Expression of CD52 antigen by dermal APC (red) compared with isotype controls (blue). Numbers indicate median fluorescence intensity ratios. Dermal T cells in the same preparation are shown for comparision. Normal skin was prepared stained and gated as described in Fig. 1.

Figure 4.

Figure 4.

Posttransplant dermal DC and macrophage chimerism. (A) Dual immunofluorescence (left) and FISH (right) of migrated dermal cells from a female recipient 40 d after transplantation. Donor-derived HLA-DR+CD14− DC and HLA-DR+CD14+ DC can be seen together with a mixture of CD3+ lymphocytes and a single recipient HLA-DR+CD14+FXIIIa+ macrophage. A small number of FXIIIa+ macrophages were seen in migratory preparations and, like the one shown, were often recipient in origin but more lightly granulated than the cells released from digested preparations. Bar, 20 µm. Dashed line indicates stitching of high-power fields. The representative example was taken from the cohort of 52 patients. (B) Dual immunofluorescence (left) and FISH (right) of remnant digested dermal cells from a male recipient 40 d after transplantation. Two donor-derived HLA-DR+CD14− dermal DC can be seen together with three recipient HLA-DR+CD14+FXIIIa+ macrophages and number of unlabeled stromal cells. Autofluorescent melanin is visible on the FISH image of the FXIIIa+ cells, confirming their identity as macrophages. Bar, 20 µm. Dashed line indicates stitching of high-power fields. The representative example was taken from the cohort of 52 patients. (C) Engraftment kinetics of dermal DC and macrophages compared with blood myeloid cells, derived from a cohort of 52 patients biopsied at 40, 100, and 365 d after transplant. Error bars show SD. *, P <0.001 compared with any other population (Mann-Whitney U test); **, P = 0.02 comparing CD14+ DC with CD14− DC (Wilcoxon rank sum test). (D) Engraftment kinetics of each cutaneous APC as a function of intensity of conditioning and prior GVHD. GVHD has a significant effect on LC engraftment both at 40 and 100 d but does not influence macrophage engraftment. Error bars show SD. *, P < 0.05 (Mann-Whitney U test).

Figure 5.

Figure 5.

Recipient dermal macrophages survive GVHD. (A) Dual immunofluorescence and FISH (insets) of a cytospin of remnant-digested dermis from a female patient with acute GVHD 40 d after transplantation. Donor-derived male DC and lymphocytes can be seen in addition to a donor eosinophil (arrows). A recipient macrophage is clearly visible. Bar, 20 µm. (B) Immunofluorescence staining of skin affected by GVHD showing CD163+ macrophages in direct contact with infiltrating CD3+ T cells (top left and bottom magnified inset). Eosinophils appear orange because of their bright AF and nonspecific staining (asterisks, a, and b). They are also highly visible in the control with secondary antibodies only (top right) and were confirmed in the section at high magnification (bottom, inset, a and b). Bar, 20 µm. The figure shows the results of one of two patients with GVHD analyzed in the same fashion. (C) Dual immunofluorescence and FISH (insets) of a cytospin of digested dermis from a female patient who had prior acute GVHD taken at 365 d after transplantation. Recipient macrophages still persist and are highly granulated. Engrafting male donor macrophages by comparison are weakly granulated. Bar, 20 µm. Dashed lines in insets indicates stitching of high power fields. (D) DNA ploidy analysis of leukocytes from normal skin. DAPI histograms are shown from a representative experiment on the left, and the means with cumulative data from five independent experiments are shown on the right. The inset shows a binucleate dermal macrophage from a male patient. Bar, 10 µm. *, P < 0.05 by Mann-Whitney U test compared with neighbor (NS, P = 0.55).

Figure 6.

Figure 6.

Cytokine production by dermal DC and macrophages. Cytokine analysis in the supernatant from 24-h culture of dermal DC, macrophages, and blood monocytes. 40,000 sorted cells were left unstimulated or treated with 10 ng/ml peptidoglycan (PGN), 10 µg/ml poly IC, and 0.1 µg/ml LPS. Data shown are the mean ± SEM of at least three independent experiments.

Figure 7.

Figure 7.

Allostimulatory properties of dermal DC and macrophages. (A) [3H]thymidine incorporation of allogeneic CD4+ T cells stimulated by CD1a+ DC or macrophages. The left and right show representative examples of six experiments in which APC were titrated against naive and memory T cells as indicated. Stimulation with anti-CD3/28 beads + naive CD4 produced 442 × 103 cpm and with anti-CD3/28 beads + memory CD4 produced 430 × 103 cpm. Results are the mean ± SEM of triplicate wells. The middle shows the cumulative results and mean of at least seven independent experiments at an APC/T cell ratio of 1:10 compared with medium alone controls (T). The stimulation index (SI) was normalized to the proliferation of naive T cells with DC. (B) Intracellular IL-17 and IFN-γ production by memory CD4+ T cells in response to DC, macrophages, anti-CD3/28 beads, or medium alone. Representative examples of four independent experiments are shown on the left, with gating for IL-17–expressing Th17 cells and IFN-γ–expressing Th1 cells. The mean and cumulative results of four independent experiments are shown on the right, expressed as the percentage of Th17, Th17/1, or Th1 cells in each. (C) [3H]thymidine incorporation of allogeneic CD8+ T cells stimulated by dermal DC or macrophages or medium alone (T). The left shows a representative example of the data at an APC/T cell ratio of 1:10. Results are the mean ± SEM of triplicate wells. The right shows the cumulative results and mean of six independent experiments. (D) IFN-γ secretion into the medium of CD8+ T cells stimulated with DC, macrophages, medium alone (T), or anti-CD3/CD28 beads (beads). Results show the mean ± SEM of six independent experiments. (E) Activation antigen expression by CD8+ T cells stimulated by DC, macrophages, anti-CD3/28 beads, or medium alone. Representative examples of the data with gating for positive cells are shown on the left. The mean and cumulative results of four independent experiments are shown on the right, expressed as the percentage of positive cells in each.

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References

    1. Steinman R.M., Lustig D.S., Cohn Z.A. 1974. Identification of a novel cell type in peripheral lymphoid organs of mice. III. Functional properties in vivo.J. Exp. Med. 139:1431–1445 - PMC - PubMed
    1. Ueno H., Klechevsky E., Morita R., Aspord C., Cao T., Matsui T., Di Pucchio T., Connolly J., Fay J.W., Pascual V., et al. 2007. Dendritic cell subsets in health and disease.Immunol. Rev. 219:118–142 - PubMed
    1. Lee S.H., Starkey P.M., Gordon S. 1985. Quantitative analysis of total macrophage content in adult mouse tissues. Immunochemical studies with monoclonal antibody F4/80.J. Exp. Med. 161:475–489 - PMC - PubMed
    1. Gordon S., Taylor P.R. 2005. Monocyte and macrophage heterogeneity.Nat. Rev. Immunol. 5:953–964 - PubMed
    1. Steinman R., Hoffman L., Pope M. 1995. Maturation and migration of cutaneous dendritic cells.J. Invest. Dermatol. 105:2S–7S - PubMed

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