TNF-alpha-dependent regulation of acute pancreatitis severity by Ly-6C(hi) monocytes in mice

Abstract

The roles of monocytes/macrophages and their mechanisms of action in the regulation of pancreatitis are poorly understood. To address these issues, we have employed genetically altered mouse strains that either express the human diphtheria toxin receptor (DTR) coupled to the CD11b promoter or have global deletion of TNF-α. Targeted, conditional depletion of monocytes/macrophages was achieved by administration of diphtheria toxin (DT) to CD11b-DTR mice. We show that in the absence of DT administration, pancreatitis is associated with an increase in pancreatic content of Ly-6C(hi) monocytes/macrophages but that this response is prevented by prior administration of DT to CD11b-DTR mice. DT administration also reduces pancreatic edema and acinar cell injury/necrosis in two dissimilar experimental models of acute pancreatitis (a secretagogue-induced model and a model elicited by retrograde pancreatic duct infusion of sodium taurocholate). In the secretagogue-elicited model, the DT-induced decrease in pancreatitis severity is reversed by adoptive transfer of purified Ly-6C(hi) monocytes harvested from non-DT-treated CD11b-DTR mice or by the transfer of purified Ly-6C(hi) monocytes harvested from TNF-α(+/+) donor mice, but it is not reversed by the transfer of Ly-6C(hi) monocytes harvested from TNF-α(-/-) donors. Our studies indicate that the Ly-6C(hi) monocyte subset regulates the severity of pancreatitis by promoting pancreatic edema and acinar cell injury/necrosis and that this phenomenon is dependent upon the expression of TNF-α by those cells. They suggest that therapies targeting Ly-6C(hi) monocytes and/or TNF-α expression by Ly-6C(hi) monocytes might prove beneficial in the prevention or treatment of acute pancreatitis.

FIGURE 1.

Effects of pancreatitis and administration of diphtheria toxin on Ly-6C^hi monocytes/macrophages in the pancreas, bone marrow, and circulating blood of CD11b-DTR mice. CD11b-DTR mice were pretreated with either vehicle (black bars) or DT (white bars) and, 16 h later, they began receiving 12 hourly injections of either saline or caerulein (caer, 50 μg/kg). They were sacrificed 12 h after the start of pancreatitis induction. Monocytes/macrophages in the pancreas (A), bone marrow (B), and circulating blood (C) were isolated and subjected to flow cytometry as described under “Results.” In each panel, the four scattergrams report cytometry results obtained after gating to select only CD45⁺, CD11b⁺, and Ly6G⁻ cells. Circumscribed areas of interest include Ly-6C^hi and 7/4⁺ cells, and the bar graph in each panel reports the quantitation of those cells. D, quantitation of Ly-6C^hi monocytes in bone marrow and blood at varying times after administration of DT to CD11b-DTR mice in the absence of pancreatitis. Results shown reflect mean ± S.D. values from four mice in each group, and asterisks indicate p < 0.05 when DT- and non-DT-treated animals in each group were compared.

FIGURE 2.

Effects of DT administration on the severity of acute pancreatitis. DT (white bars) or saline (black bars) was given to CD11b-DTR mice, and pancreatitis was induced 16 h later by either administration of caerulein (A) or retrograde intraductal infusion of sodium taurocholate (Na-taurocholate) (B). Twenty-four hours after the start of pancreatitis induction, the animals were sacrificed, and the severity of pancreatitis was determined as described under “Results.” In other studies (C), the interval between DT administration and the start of caerulein (caer) administration was varied, as indicated, from 1 to 7 days. Results shown reflect mean ± S.D. values obtained from 4–9 animals in each group. Bars in photomicrographs indicate 100 μm. Asterisks denote p < 0.05 when DT and saline-treated animals were compared. NaT, sodium taurocholate.

FIGURE 3.

Effects of adoptive transfer on pancreatitis severity after DT administration. CD11b-DTR mice were given either saline (black bars) or DT (white and gray bars). Sixteen hours later, pancreatitis was induced by the hourly (12×) administration of caerulein. During the second hour of caerulein administration, adoptive transfer (10⁶ cells/transfer) was performed as described under “Results” (gray bars). Unless otherwise stated, the transferred cells consisted of either PBMCs or BMCs that were prepared as described under “Results,” and they were infused intravenously. Recipient mice received (a) PBMCs (PBMC), (b) PBMCs given i.p. (PBMC ip), (c) PBMCs harvested from donors pretreated with DT (PBMC DT), (d) BMCs (BMC), (e) BMCs depleted of Ly-6C⁺ cells (BMC-Ly-6C), (f) BMCs enriched in Ly-6C⁺ monocytes by positive selection (Ly-6C+), or (g) purified Ly-6C^hi cells obtained from BMCs by FACS (Ly-6Chi). The animals were sacrificed 24 h after the start of caerulein administration, and pancreatic edema and acinar cell injury/necrosis were quantitated as described under “Results.” Results reflect mean ± S.D. values obtained from 3–9 mice per group. Asterisks denote p < 0.05 when compared with results obtained from mice not treated with DT. NS, not significant.

FIGURE 4.

Effect of pancreatitis, DT administration, and adoptive transfer on the content of Ly-6C^hi monocytes/macrophages in the pancreas. CD11b-DTR mice were given either saline (black bars) or DT (white and gray bars). Sixteen hours later, pancreatitis was induced by the hourly (12×) administration of caerulein (caer). During the second hour of caerulein administration, randomly selected animals were adoptively transferred (ad tr) with BMC or PBMC (10⁶ cells/transfer) as described under “Results” (gray bars). Mice were sacrificed 24 h after the start of pancreatitis induction, and the pancreatic content of Ly-6C^hi cells was quantitated by flow cytometry as described under “Results.” Results reflect mean ± S.D. values obtained from 3–9 mice per group. Asterisks denote p < 0.05 when DT-treated mice were compared with mice not given DT. NS, not significant.

FIGURE 5.

Effects of TNF-α deletion or expression on pancreatitis severity. A, TNF-α^+/+ and TNF-α^−/− mice (both of the FVB/N strain) underwent induction of pancreatitis by repeated (12×) hourly administration of caerulein. During the second hour of caerulein administration, randomly selected TNF-α^−/− mice were adoptively transferred with Ly-6C^hiLy6G⁻ monocytes (10⁶ cells/mouse) harvested from either TNF-α^+/+ or TNF-α^−/− donor mice as described under “Results” (gray bars). Pancreatitis severity was evaluated 24 h after the start of pancreatitis induction by quantitation of pancreatic edema and acinar cell injury/necrosis as described under “Results.” B, FVB/N CD11b-DTR mice were given saline (black bars) or DT (white and gray bars) and, 16 h later, pancreatitis was induced by repeated (12×) hourly administration of caerulein. During the second hour of caerulein administration, randomly selected, DT-treated mice were adoptively transferred with Ly-6C^hiLy6G⁻ monocytes (10⁶ cells/mouse) harvested from either TNF-α^+/+ or TNF-α^−/− donor mice as described under “Results” (gray bars). Pancreatitis severity was evaluated 24 h after the start of pancreatitis induction by quantitation of pancreatic edema and acinar cell injury/necrosis as described under “Results.” Results reflect mean ± S.D. values obtained from three or more mice per group. Asterisks denote p < 0.05 when DT-treated mice were compared with mice not given DT. NS, not significant.