IL-4 and TGF-beta 1 counterbalance one another while regulating mast cell homeostasis

Abstract

Mast cell responses can be altered by cytokines, including those secreted by Th2 and regulatory T cells (Treg). Given the important role of mast cells in Th2-mediated inflammation and recent demonstrations of Treg-mast cell interactions, we examined the ability of IL-4 and TGF-beta1 to regulate mast cell homeostasis. Using in vitro and in vivo studies of mouse and human mast cells, we demonstrate that IL-4 suppresses TGF-beta1 receptor expression and signaling, and vice versa. In vitro studies demonstrated that IL-4 and TGF-beta1 had balancing effects on mast cell survival, migration, and FcepsilonRI expression, with each cytokine cancelling the effects of the other. However, in vivo analysis of peritoneal inflammation during Nippostrongylus brasiliensis infection in mice revealed a dominant suppressive function for TGF-beta1. These data support the existence of a cytokine network involving the Th2 cytokine IL-4 and the Treg cytokine TGF-beta1 that can regulate mast cell homeostasis. Dysregulation of this balance may impact allergic disease and be amenable to targeted therapy.

FIGURE 1

IL-4 suppresses TGF receptor expression and signaling. A, Mouse BMMCs were cultured for 4 d in the presence or absence of IL-4, and TGF-βR1 and -2 expression was assessed by flow cytometry after fixation and permeabilization. Data shown are the average of four samples, represented as geometric mean fluorescent intensities ±SE. B, Example of TGF-βR2 staining performed on cells cultured as in A. C and D, WT and Stat6-deficient KO BMMC were cultured as in A, and expression of TGF-βR1 or TGF-βR2 mRNA was assessed by RNase protection assay on day 4. Data shown are the mean percent of loading controls (L32 and GAPDH) ± SE from five samples. *p <0.05; ***p <0.001 based on t test comparing cells receiving IL-4 or not.

FIGURE 2

IL-4 exposure inhibits subsequent TGF effects on mast cells. A, Cells were cultured as in Fig. 1, and total cell lysates were subjected to Western blotting to detect total Smad2. Right-hand side shows mean ±SE Smad2:actin ratios for six samples. B, BMMCs were cultured as in Fig. 1, then stimulated for 0 or 5 min with TGF-β1. Total cell lysates were subjected to Western blotting to detect phosphorylated pSmad2. Membrane was reprobed for actin to show protein loading. Right-hand side shows fold pSmad2 levels compared with unstimulated cells cultured in IL-3 alone. Data shown are mean ± SE of three samples. ***p <0.001 by t test. C, BMMCs were precultured for 4 d in IL-3 ±IL-4, then replated in the same media ±TGF for 6 d. Viable cell numbers were assessed by propidium iodide staining, as described in Materials and Methods. Data shown are mean ±SE of four samples/group. ***p <0.001 based on t test of samples ±IL-4. D, BMMCs were cultured as in C, then tested for TGF-induced migration, as described in Materials and Methods. Fold migration is relative to control cells receiving media alone as a migration stimulus. Data shown are mean ±SE of three samples/group. **p <0.02 based on t test of samples ±IL-4.

FIGURE 3

TGF suppresses IL-4 receptor expression on mouse mast cells. BMMCs were cultured for 4 d in IL-3 ± TGF. In A, surface IL-4Rα was measured by flow cytometry, with an example histogram on the left and summary of four sample sets on the right. B, Shows an example Western blot of IL-4Rα on the left, and a summary of nine Western blots, normalized to actin expression and compared with control IL-3 sample. C, Shows an example of intracellular staining for γc expression on the left, and a summary of four sample sets on the right. **p < 0.01; ***p < 0.001 based on t test of samples ± TGF.

FIGURE 4

TGF exposure inhibits subsequent IL-4 signaling. BMMCs were cultured for 4 d in IL-3 ± TGF, then washed and restimulated with IL-4 for the indicated times. A, Shows an example Western blot of ty-rosine phosphorylated Stat6, total Stat6, and actin on the left, and a summary of four Western blots analyzed by densitometry on the right. Note that phosphorylated Stat6 migrates as a single detectable band, whereas total Stat6 is detected as 2 cleaved bands. B, Human mast cells were cultured as in A, using SCF-containing media ± TGF, then washed and restimulated with IL-13 for 10 min. Total cell lysates were assessed for tyrosine phosphorylated Stat6 levels by Western blotting. The same membrane was stripped and reprobed to detect total Stat6 and actin. C, BMMCs were cultured as in A, then washed and assessed for IL-4–induced migration as described in Materials and Methods. Fold migration is relative to control cells receiving media lacking IL-4 as a migration stimulus in each group. Data shown are mean ± SE of 12 samples/group. For A and C, *p < 0.05; **p < 0.01 based on ANOVA analysis, comparing all groups to control sample in each group (i.e., first bar in each graph).

FIGURE 5

IL-4 and TGF have opposite effects on mast cell proliferation and survival in vitro. A, BMMCs were cultured in the indicated cytokines for 4 d, and surface expression of IL-3Rβ was assessed by flow cytometry. Data are mean and SEM of seven samples. B, WT or Stat6-deficient KO BMMC were cultured at a starting concentration of 50 × 10⁴/ml for 4 d with the indicated cytokines in the presence or absence of the PI3K inhibitor, LY294002. Live cell numbers were calculated by trypan blue counting. Data shown are mean ± SE of four sets of paired samples, from one of two experiments. *p < 0.05; **p < 0.01; ***p < 0.001; NS compared with control culture in IL-3 alone for each set of samples, based on ANOVA analysis.

FIGURE 6

IL-4 and TGF exert opposing effects during N. brasiliensis infection. Mice were infected with N. brasiliensis, then injected with IL-4 and/or TGF on days 3–6, as described in Materials and Methods. A–F, Shows analysis of peritoneal lavage fluid harvested on day 7, from which cell numbers were determined by trypan blue staining and flow cytometry. G, Shows mast cell FcεRI expression (geometric mean fluorescence intensity), as assessed by flow cytometry after gating on KIT⁺ cells. Data shown are mean ± SE of three to six mice per sample set. H, Peritoneal cells were harvested by lavage from naive mice and cultured for 4 d in the indicated cytokines. Peritoneal mast cells were analyzed for FcεRI expression after gating on Kit⁺ population. For A–G, ANOVA analysis compared all groups to infected, PBS-injected mice (i.e., “PBS Control” bar for each graph). For H, all groups are compared with IL-3 sample. *p < 0.05; **p < 0.01; ***p < 0.001.

FIGURE 7

IL-4 and TGF have opposing effects on human mast cells. A, Human skin-derived mast cells from two donors were precultured for 4 d in SCF-containing media (“Media”) ± IL-4 or TGF, then washed and assessed for migration in response to TGF-β1 or IL-4 as described in Materials and Methods. Fold migration was calculated compared with SCF-containing media alone, which is omitted for clarity. Data shown are mean ± SE of triplicate samples from each donor. B, Human skin mast cells from two donors (distinct from donors in A) were cultured in media ± IL-4 and/or TGF-β1 for 3 d, and surface FcεRI expression was measured by flow cytometry. Data shown are mean ± SE of triplicate samples from each donor. *p < 0.05; **p < 0.01; ***p < 0.001 based on ANOVA analysis comparing all samples to media control for that group (i.e., first bar in each donor sample set).