PROGASTRIN INHIBITS MONOCYTE/MACROPHAGE DIFFERENTIATION AND REPRESS THE ALTERNATIVE ACTIVATION OF MATURE MACROPHAGES

D ORTIZ-MASIA1, C HERNANDEZ3, AJ PEREZ-SOTO2, J COSIN-ROGER2, A ALVAREZ2,1, MD BARRACHINA2,1, S CALATAYUD2,1. 1CIBERehd, Gastrointestinal inflammation and Motility, Spain, 2Universidad de Valencia, Pharmacology, Spain, 3Fundacion Hospital Dr Peset, Pharmacology, Spain

Most colon cancers express the gastrin gene and synthesize significant quantities of the immature peptide progastrin. Progastrin is known by its proliferative action on cancer cells and the only receptor identified as mediator of this effect is annexin II (Cancer Lett 2007, 252:19). This protein is highly expressed on the surface of macrophages and participates in many facets of macrophage biology. Macrophages are a significant component of tumors, where they can fight against cancerous cells (classically activated macrophages, pro-inflammatory, M1) or promote tumor growth (alternatively activated macrophages, tolerogenic, M2) (Nat Immunol 2010: 10:889). Our hypothesis is that the locally produced progastrin in colonic tumors can influence the function of infiltrated macrophages and, in this way, modulate the immune response to disease.

The aim of the present study was to analyze whether progastrin modulates the differentiation of monocytes into macrophages as well as the activation of mature macrophages.

Methods. Human peripheral blood monocytes were cultured for 6 days in the presence of MCSF to obtain macrophages. Control mature cells were activated with E.coli LPS (100 ng/ml) plus IFNγ (20ng/ml, 24h) to induce M1 macrophages, or with IL4 (20ng/ml, 48h) to induce M2 macrophages. The maturation process as well as the activation treatments were carried out in the presence of progastrin (10-11-10-8M) or its vehicle.

In these cells, we analyzed the expression of the protein LC3-II (western blot) as a marker of autophagy, process recently involved in the monocyte/macrophage differentiation (Blood 2012, 119:2895). Secretion of the cytokines IL12 (Th1) and IL10 (Th2, anti-inflammatory) was evaluated by ELISA. The expression of the surface molecules CD86 (M1 marker) and CD206 (absent in monocytes, M2 marker) was analyzed by static cytometry (ScanR).

Results. Macrophages obtained by culturing monocytes in the presence of progastrin for 3 days showed a reduced expression of LC3-II (PG 10-8M: 31±3% reduction vs veh, p<0.05). This change was accompanied by a reduction in IL10 secretion (PG 10-8M: 66±12% reduction vs veh at day 3, 68±13% reduction vs veh at day 6, p<0.05 both) but normal levels of IL12. Progastrin also reduced the expression of CD206 (PG 10-8M: 16±2% reduction vs veh, p<0.05) without changes in CD86 expression at six days.

IL4 treatment induced a significant increase in CD206 expression, a non-significant increase in IL12 secretion and did not modify IL10 production. In these conditions, progastrin significantly reduced the expression of CD206 (PG 10-8M: 23±2% reduction vs veh, p<0.05) and increased IL12 secretion to levels significantly higher than those observed in control cells (control: 0.2±0.2; IL4: 3.8±0.8; IL4 + PG 10-8M: 6.3±2.5* pg/ml, *p<0.05 vs control), while secretion of IL10 remained unchanged. Treatment with LPS plus IFNγ increased the expression of CD86, and the secretion of both IL12 and IL10, but progastrin did not modify any of these parameters.

Our results suggest that progastrin can influence the monocyte/macrophage differentiation process, an effect that probably implies a defective autophagy. Moreover, this peptide inhibits the derivation towards an M2 phenotype, an action that, exerted on tumor associated macrophages, would imply a beneficial effect.