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. 2013 Aug 5;202(3):563-77.
doi: 10.1083/jcb.201301001.

Macrophage-secreted cytokines drive pancreatic acinar-to-ductal metaplasia through NF-κB and MMPs

Geou-Yarh Liou  1 , Heike Döppler, Brian Necela, Murli Krishna, Howard C Crawford, Massimo Raimondo, Peter Storz
Affiliations

Macrophage-secreted cytokines drive pancreatic acinar-to-ductal metaplasia through NF-κB and MMPs

Geou-Yarh Liou et al. J Cell Biol. .

Abstract

In response to inflammation, pancreatic acinar cells can undergo acinar-to-ductal metaplasia (ADM), a reprogramming event that induces transdifferentiation to a ductlike phenotype and, in the context of additional oncogenic stimulation, contributes to development of pancreatic cancer. The signaling mechanisms underlying pancreatitis-inducing ADM are largely undefined. Our results provide evidence that macrophages infiltrating the pancreas drive this transdifferentiation process. We identify the macrophage-secreted inflammatory cytokines RANTES and tumor necrosis factor α (TNF) as mediators of such signaling. Both RANTES and TNF induce ADM through activation of nuclear factor κB and its target genes involved in regulating survival, proliferation, and degradation of extracellular matrix. In particular, we identify matrix metalloproteinases (MMPs) as targets that drive ADM and provide in vivo data suggesting that MMP inhibitors may be efficiently applied to block pancreatitis-induced ADM in therapy.

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Figures

Figure 1.
Figure 1.
Depletion of macrophages blocks pancreatic ADM. (A) Samples of human pancreatitis or adjacent normal tissue were stained by immunofluorescence for macrophages (EMR1), amylase, CK-19 (gray in A6), and DAPI as indicated. Bars, 20 µm. (B) FVB mice were treated with control vehicle, GdCl3, caerulein, or a combination, as indicated (detailed method in Materials and methods section). Pancreata were harvested and IHC stained with H&E or the macrophage marker F4/80. Boxes in A and B indicate enlarged regions. (C) Quantitation of ADM events per field of n = 5 samples from B. The asterisk indicates statistical significance as determined by the Student’s t test. Error bars show means ± SD. (D, top row) Immunofluorescence staining of F4/80, amylase, and DAPI in samples from FVB mice treated with control vehicle, GdCl3, caerulein, or a combination, as indicated (detailed method in Materials and methods section). (second row) F4/80 channel alone in white. (third row) Immunofluorescence staining of CK-19, amylase, and DAPI. (fourth row) CK-19 channel and DAPI. Bars, 25 µm. See also Fig. S1.
Figure 2.
Figure 2.
Macrophages induce ADM of pancreatic acinar cells. (A) Freshly isolated, activated mouse primary macrophages and mouse primary acinar cells were co-cultured in 3D collagen explant culture. At day 5, ADM events per well were quantified by counting. (B) Raw 264.7 and freshly isolated mouse primary acinar cells were in vivo labeled with Vybrant dyes (pseudocolors: blue, macrophages [M]; red, acinar cells [AC]) and co-cultured in 3D collagen explant culture. At day 6, co-cultures were analyzed for duct (D) formation by fluorescence microscopy (bar, 50 µm). See also Fig. S2 A. (C) Primary pancreatic acinar cells were isolated and embedded in collagen in µ-Slides (Ibidi) for cell co-cultivation (magenta area in scheme). Similarly, Raw 264.7 cells (cyan areas in scheme) were embedded in collagen (or collagen alone as a control). Cells in the collagen matrix were overlayed with Waymouth media. ADM events in the acinar cell area were determined by counting ducts. (D) Primary mouse pancreatic acinar cells were isolated and cultivated in 3D collagen explant culture in presence of Raw 264.7–, WR19M.1- or primary mouse macrophage-conditioned media, and ADM events per well were determined (graph). (E) Photos show ducts obtained (at day 5) after treatment with Raw 264.7–conditioned media as compared with untreated acinar cells and cells treated with 50 ng/ml TGF-α. Bars, 100 µm. See also Fig. S2 B. (F) Primary mouse pancreatic acinar cells were isolated and treated with Raw 264.7–conditioned media to induce ADM. At day 2, cells were isolated from the 3D collagen explant culture, and quantitative real-time PCR for indicated markers of ADM events was performed. (G) Primary mouse pancreatic acinar cells were isolated and treated with Raw 264.7–conditioned media to induce ADM. Cells were isolated from the 3D collagen explant culture, and cell lysates were analyzed for expression of the ductal marker CK-19 or the acinar marker amylase by Western blotting. Silver staining served as a loading control. (H) Primary mouse pancreatic acinar cells were isolated and treated with Raw 264.7–conditioned media to induce ADM in presence of 3 µg/ml TGF-α neutralizing antibody (mTGFα-NAB) or 1 µM Erlotinib as indicated. Treatment with 50 ng/ml TGF-α served as a comparison to conditioned media. At day 5, ADM events per well were quantified by counting. The gray line represents onefold ADM events. In the experiments depicted in A, C, D, F, and H, bar graphs show means ± SD of n = 3 experiments. The asterisk indicates statistical significance as determined by the Student’s t test. All experiments depicted have been repeated with similar results at least three times.
Figure 3.
Figure 3.
Macrophage-secreted factors RANTES and TNF mediate ADM. (A) Conditioned media from Raw 264.7, WR19M.1, activated primary mouse macrophages, or control media (bottom) were subjected to a mouse cytokine profiler array allowing the detection of 40 different cytokines (see table in Fig. S3 A for cytokine identity and position on the array; A1/2, A23/24, and F1/2 contain positive controls, and F23/F24 contains negative controls). (B) Mouse primary acinar cells were co-cultured in 3D collagen explant culture in the presence of the cytokines (G-CSF, IP-10, JE, MIP-1α, MIP-1β, MIP2, RANTES, or TNF; all at 50 ng/ml) that were specifically identified in conditioned media from macrophages. Shown is a representative picture of each condition showing either acinar or ADM clusters. (C) Primary mouse pancreatic acinar cells were isolated and treated with Raw 264.7–conditioned media to induce ADM in the presence of neutralizing antibodies (NAB) for 0.5 µg/ml mRANTES and 3 µg/ml mTNF-α as indicated. At day 5, ADM events per well were quantified by counting. The single asterisk indicates statistical significance as compared with control determined by the Student’s t test. Double asterisks indicate statistical significance as compared with control and as compared with Raw 264.7–conditioned medium–induced ADM as determined by the Student’s t test. Bar graph shows means ± SD of n = 3 experiments. (D) Immunofluorescence staining of amylase, RANTES, and DAPI in human pancreas tissues containing a pancreatitis-affected area (top row) and adjacent normal area (bottom row). (left, D1 and D4) Amylase + DAPI; (middle, D2 and D5) RANTES; (right, D3 and D6) merged images containing amylase, DAPI, and RANTES. (E) Immunofluorescence staining of amylase, TNF, and DAPI in human pancreas tissues containing areas affected by pancreatitis (top row) or adjacent normal area (bottom row). (left, E1 and E4) Amylase + DAPI; (middle, E2 and E5) TNF; (right, E3 and E6) merged images containing amylase, TNF, and DAPI. See also Fig. S3 B. All experiments depicted have been repeated with similar results at least three times. Bars: (B) 200 µm; (D and E) 20 µm.
Figure 4.
Figure 4.
RANTES and TNF induce acinar cell transdifferentiation through NF-κB. (A) Primary mouse pancreatic acinar cells were isolated and infected with adenovirus carrying an NF-κB–luciferase reporter. Cells were treated with control or Raw 264.7–conditioned media for 24 and 48 h. NF-κB promoter activity was determined by measuring luciferase activity. NF-κB induction as compared with control (set to 100%) is shown. The asterisk indicates statistical significance as compared with control. (B) Primary mouse pancreatic acinar cells were isolated and treated with Raw 264.7–conditioned media in the presence of 25 µM SC514 or 1 µM BMS345541 as indicated. ADM events per well were quantified by counting ducts formed. (C) Primary mouse pancreatic acinar cells were isolated and treated with Raw 264.7–conditioned media, 50 ng/ml TNF, or 50 ng/ml RANTES as indicated for 24 h. Cells were lysed and analyzed by Western blotting for IκBα degradation using an α-IκBα antibody. (D) Primary mouse pancreatic acinar cells were isolated and infected with control virus or adenovirus to express superdominant IκBα (IκBα.SD). Cells were then cultivated in 3D collagen explant culture in presence of Raw 264.7–conditioned media or control media as indicated. ADM events per well were quantified by counting. See also Fig. S4. (E) Primary mouse pancreatic acinar cells were isolated and infected with control virus or adenovirus to express superdominant IκBα (IκBα.SD). Cells were then cultivated in 3D collagen explant culture in presence of 50 ng/ml TNF or 50 ng/ml RANTES as indicated. ADM events per well were quantified by counting. (F) Primary mouse pancreatic acinar cells were isolated and infected with control virus or adenovirus to express NF-κB1/p105. Cells were then cultivated in 3D collagen explant culture. Representative photographs of the cells in 3D culture are shown (left side), and ADM events per well were quantified by counting (right side). Bars, 200 µm. (G) Primary mouse pancreatic acinar cells infected with control virus or adenovirus to express NF-κB1/p105 were isolated from 3D collagen explant culture at day 3. Quantitative PCR for the indicated markers of ADM events was performed. Dotted line represents onefold expression. In the experiments depicted in A, B, and D–G, bar graphs show means ± SD of n = 3 experiments. The asterisks indicate statistical significance as determined by the Student’s t test (single asterisks show statistical significance relative to the control; double asterisks show statistical significance relative to the stimulus). All experiments depicted have been repeated with similar results at least three times. CM, conditioned media.
Figure 5.
Figure 5.
NF-κB drives acinar cell transdifferentiation through MMP-9. (A) Primary mouse pancreatic acinar cells that were infected with control virus or adenovirus to express NF-κB1/p105 were isolated at day 6 from 3D collagen explant culture. Quantitative real-time PCR for the indicated MMPs was performed. Dotted line represents onefold expression. Bar graphs show means ± SD of n = 3 experiments. (B) Gelatin zymography with supernatants from primary mouse pancreatic acinar cells that were infected with control virus or adenovirus to express NF-κB1/p105 and underwent ADM in 3D collagen explant culture. The arrow indicates active MMP-9. (C) Primary mouse pancreatic acinar cells were isolated and cultivated in 3D collagen explant culture in presence of recombinant MMP-9 (1 and 5 ng/ml). (left) At day 5, ADM events per well were quantified by counting. (right) Representative photographs of the cells in 3D culture are shown. Bars, 100 µm. (D) Primary mouse pancreatic acinar cells were isolated and cultivated in 3D collagen explant culture in presence of Raw 264.7–conditioned media and the MMP inhibitor GM6001 (2 µM) as indicated. At day 5, ADM events per well were quantified by counting. (E) Primary mouse pancreatic acinar cells were isolated and cultivated in 3D collagen explant culture in the presence of the MMP inhibitor GM6001 (2 µM), 50 ng/ml TNF, or 50 ng/ml RANTES as indicated. At day 5, ADM events per well were quantified by counting. (F) Primary mouse pancreatic acinar cells were isolated, infected with control virus or adenovirus to express NF-κB1/p105, and cultivated in 3D collagen explant culture in the presence of the MMP inhibitor GM6001 (2 µM). (top) At day 7, ADM events per well were quantified by counting. (bottom) Representative photographs of the cells in 3D culture are shown. Bars, 200 µm. In the experiments depicted in A and C–F, bar graphs show means ± SD of n = 3 experiments. The asterisks indicate statistical significance as determined by the Student’s t test (single asterisks show statistical significance relative to the control; double asterisks show statistical significance relative to the stimulus). All experiments depicted have been repeated with similar results at least three times.
Figure 6.
Figure 6.
MMPs contribute to ADM in vivo. (A) FVB mice were treated with control vehicle, GdCl3, caerulein, or a combination, as indicated. Pancreata were harvested and IHC stained for MMP-9 expression. (B) FVB mice were treated with control vehicle, GM6001, caerulein, or a combination, as indicated (detailed method in the Materials and methods section). Pancreata were harvested and IHC stained with H&E or Alcian blue. Boxes show the sample at a higher magnification. (C) Quantitation of ADM events per field of n = 5 samples from B. Bar graphs show means ± SD. The asterisk indicates statistical significance as determined by the Student’s t test. (D) MMP-9 expression in human pancreas tissues containing areas that are affected by pancreatitis and the adjacent normal regions. (E) Schematic of the proposed signaling events induced by macrophages after pancreatic inflammation. Our data suggest that macrophage-secreted inflammatory cytokines, such as RANTES and TNF, induce NF-κB in pancreatic acinar cells and that the target genes for this transcription factor can drive ADM. Bars, 100 µm.
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