Myeloid cells migrate in response to IL-24

Abstract

IL-24 (melanoma differentiation associated gene 7 product) is a member of the IL-10 cytokine family that has been reported to possess anti-tumor activity. IL-24 is produced by immune tissues and its expression can be induced in human peripheral blood mononuclear cells by pathogen-associated molecules. While immune cells are known to produce IL-24, the response of immune cells to IL-24 is unclear. Using recombinant human IL-24, we demonstrated that IL-24 induces human monocyte and neutrophil migration, in vitro. An in vivo chemotaxis model showed that IL-24 attracted CD11b positive myeloid cells. To further characterize the chemotactic IL-24 response and type(s) of receptor(s) utilized by IL-24, we treated monocytes with signaling pathway inhibitors. IL-24-induced migration was reduced by pertussis toxin treatment, thus implicating G-protein coupled receptors in this process. Additionally, MEK and JAK inhibitors markedly decreased monocyte migration toward IL-24. These results suggest that IL-24 activates several signaling cascades in immune cells eliciting migration of myeloid cells, which may contribute to the known anti-cancer effects of IL-24.

Figure 1. IL-24 induced human monocyte and neutrophil migration – In vitro

Human monocytes were assayed for chemotactic activity using a micro-Boyden chamber. Recombinant human IL-24 was acquired from R&D Systems and diluted in chemotaxis media. Chemotactic Index is shown on the y-axis and is relative to spontaneous migration, mean and sem are reported. Migration of human monocytes are shown with right hatched and black bars, while migration of human neutrophils is shown with gray bars. * indicate a p-value < 0.001 relative to spontaneous migration, determined by unpaired T-test. N>6

Figure 2. IL-24 induced leukocyte migration - in vivo

Recombinant IL-24 in HBSS was injected into air pouches formed on the back of C57BL/6 mice. Twenty-four hours later, cells were recovered from the air pouches and counted. Mean cell numbers in millions ± sem are reported for each treatment condition, with the control being HBSS containing no chemoattractant. * p= 0.01 student-T two tailed compared to control ** p=0.003 student-T two tailed compared to control. N≥4.

Figure 3. FACS analysis of in vivo recruited leukocytes

A. Mean proportion or percentage of all recruited leukocytes staining positive for both CD45 and CD3 is shown, along with ± sem, compared to different eliciting agents injected into the air pouch N≥4. B. Representative dot plot graphic of recruited CD45 gated cells stained for CD3 on the x-axis or CD11b on the y-axis. Percentage of CD45 gated cells staining positive for either CD3 or CD11b are reported in the inset. C. Mean percentage of recruited CD11b positive cells also staining positive for F4/80 is shown ± sem, compared to different eliciting agents injected into the air pouch N≥4 D. Representative histograms of CD11b+ cells that are also F4/80 positive. Percentage positive (to the right) or negative (to the left) is shown by inset numbers. The shaded histogram shows an isotype control for F4/80. *p≤0.024 compared to control HBSS injected into the air pouch

Figure 4. IL-24-induced human monocyte migration can be inhibited

A. Pertussis toxin pretreatment inhibits IL-24 induced monocyte chemotaxis. Chemotaxis to media, CCL2 or IL-24 is shown in hatched bars. The chemotaxis of monocytes pretreated for 30 minutes with 100ng/ml pertussis toxin is shown in solid black bars. * indicates increases in migration with p-values >0.001 compared to media control # indicates decreases in migration with p-values >0.01 compared to no pertussis toxin at the same concentration of chemoattractant. B: Pretreatment of monocytes with U0126 inhibits IL-24-induced monocyte chemotaxis. Human monocytes were pretreated for 30 minutes with 50μM U0126 (MEK inhibitor) prior to being placed in a micro-Boyden chamber. Media alone is shown in white bars. IL-24 is shown with black bars. U0126 treatment is indicted in hatched bars. * indicates increases in migration with p-values >0.001 compared to media control # indicates decreases in migration with p-values > 0.001 compared to no U0126 treatment C: Pretreatment of monocytes with AG 490 inhibits IL-24-induced monocyte chemotaxis Human monocytes were incubated with 50 μM AG 490 (JAK inhibitor) for 30 minutes prior to being placed in a chemotaxis chamber. Media is shown with white bars, CCL2 (100 ng/ml) is shown with gray bars, IL-24 is shown with black bars. AG-490 treated cells are shown with hatched bars. * indicates increases in migration with p-values >0.001 compared to media control # indicates decreases in migration with p-values > 0.001 compared to no AG-490 treatment. N > 6