Interleukin-4 and interferon-gamma synergistically increase secretory component gene expression, but are additive in stimulating secretory immunoglobulin A release by Calu-3 airway epithelial cells

Abstract

Interleukin-4 (IL-4) and interferon-gamma (IFN-gamma) synergize to express polymeric immunoglobulin receptor (pIgR) but their combined effect, and that of IL-4 alone, on secretory immunoglobulin A (sIgA) release is unknown. Recently, we have developed an airway epithelial cell model that allows assessment of the integrated effect of a stimulus on pIgR gene and protein expression and sIgA release. With this model we show here that IL-4 and IFN-gamma dose-dependently increased pIgR mRNA and protein expression, and sIgA release. IFN-gamma and IL-4 induced similar maximal expression of pIgR, but IFN-gamma enhanced sIgA release more than IL-4. When added together, IL-4 and IFN-gamma synergistically increased pIgR mRNA and protein expression, but sIgA release was stimulated in an additive manner. Thus, IL-4 and IFN-gamma may be implicated in the increase of sIgA levels as found in mucosal inflammatory diseases. In addition, our results indicate that transport and release of empty pIgR is subject to regulatory mechanisms different from those of pIgR with bound dimeric IgA.

Figure 1

IL-4 and IFN-γ dose-dependently increase release of free SC. Confluent Calu-3 monolayers were grown on 12-mm culture inserts. Cells were incubated with (a) IL-4 (0–1000 U/ml) or (b) IFN-γ (0–800 U/ml), for 48 hr. Apical supernatants were harvested for assessment of free SC. Data are expressed as ng SC/insert (mean±SD, n = 3; IL-4 1000 U/ml was tested in duplicate). The experiment shown is representative of three independent experiments but 1000 U/ml of IL-4 has been tested in two independent experiments only. (a) Linear regression analysis indicated a highly significant (P < 0·0031) correlation between increasing IL-4 concentrations and apical free SC release (correlation coefficient: r = 0·77), indicating a linear dose-dependency. (b) *Significantly higher than unstimulated condition (P < 0·05); ***significantly higher than unstimulated condition (P < 0·001).

Figure 2

IL-4 and IFN-γ dose-dependently increase sIgA release. Cells were incubated with IL-4 (a) or IFN-γ (b), as described in Fig. 1. After 48 hr, medium was refreshed and 50 μg dIgA was added to the basolateral compartment. Transcytosis was allowed to proceed for an additional 24 hr. Supernatants were collected for assessment of sIgA. Data are expressed as ng sIgA/insert (mean±SD, n = 3). The experiment shown is representative of three independent experiments. ***Significantly higher than parallel unstimulated condition (P < 0·001). (a) * and **Significantly lower (P < 0·05 and P < 0·01, respectively) than cultures stimulated with 100 U/ml of IL-4. (b) * and **Significantly higher (P < 0·05 and P < 0·01, respectively) than cultures stimulated with 50 U/ml of IFN-γ.

Figure 3

IL-4 plus IFN-γ synergistically increase free SC release and sIgA release. Cells were incubated with IFN-γ (0–800 U/ml) in the presence of 100 U/ml of IL-4, for 48 hr. Medium was refreshed and apical samples were collected for assessment of free SC. Then, 50 μg dIgA was added to the fresh medium in the basolateral compartment, and transcytosis was allowed to proceed for another 24 hr. Then, apical supernatants were collected for assessment of sIgA. Data are expressed as ng SC/insert and ng sIgA/insert (mean±SD; n = 3). The experiment shown is representative of two independent experiments. Linear regression analysis indicated a highly significant correlation (P < 0·0016) between IL-4-stimulated (100 U/ml) cultures incubated with increasing concentrations IFN-γ and apical sIgA release (correlation coefficient: r = 0·83), indicating a linear dose-dependency. (a) *Significantly different from unstimulated condition (P < 0·05), and ***Significantly different from parallel IFN-γ-stimulated condition (P < 0·001). (b) * and ***Significantly different from parallel IFN-γ-stimulated condition (P < 0·05 and P < 0·001, respectively).

Figure 4

IL-4 plus IFN-γ synergistically induce pIgR mRNA accumulation. Cells were grown on six-well inserts and incubated with IL-4 (100 U/ml), IFN-γ (200 U/ml) or with both cytokines, for 48 hr. Then, RNA was isolated and processed as described in the Material and Methods. (a) Phosphor-image of Northern blot hybridized with radiolabelled human SC and rat GAPDH-probes. Lane 1, unstimulated; lane 2, IFN-γ; lane 3, IL-4; and lane 4, IFN-γ+IL-4. (b) Densitometric quantification of phosphor-image signals in (a); the ratio of SC message to GAPDH message was used to correct for different loading of RNA. Data are expressed as fold increase relative to unstimulated condition, which is arbitrarily set at 1·0. The experiment shown is representative of three independent experiments.