IL-6Cooperates With G-CSF To Induce Protumor Function Of Neutrophils By Modulating Their Degranulation Function

Polymorphonuclear leukocytes (PMNs or neutrophils) make up a significant portion of the inflammatory cell infiltrate found in a wide variety of human cancers and murine models. They are known to have anti-tumor potential. However, in recent years the tumor-promoting effect of neutrophils has been well demonstrated. So far it remains unclear what causes the conversion of neutrophil function from tumor suppressive to tumor promotional. Here we report that the conversion of murine neutrophil function occurs in bone marrow, and that IL-6cooperation with G-CSF is required for causing this conversion. IL-6cooperated with G-CSF to modulate neutrophils in bone marrow, altering the activation potential of signaling pathways in neutrophils resulting in the attenuating of degranulationg ability of PMN. Co-stimulation with G-CSF and IL-6attenuated the activation of and increased STAT3expression in neutrophils. In one hand, the attenuating of the PI3K and p38MAPK pathways caused inefficient inducing degranulation of TRAIL, MPO and NE. On the other hand, the heigher activation of STAT3pathway downregulated the Rab27a resulting in the decreasing of spontaneous degranulation. Taken together, these results demonstrate that G-CSF and IL-6, despite their well-known physiological functions, could modulate the activation potential of signaling pathways in neutrophils, resulting in the production or release of the above-mentioned factors in a way that favors tumor growth. Objective:To explore whether the function of neutrophils could be modulated before they entered tumor milieu and what factors cause this convertion.Methods:we first tested the effect of bone marrow neutrophils on tumor growth in co-inoculation test. To clarify the roles of G-CSF and IL-6, we evaluated their effect on the function of bone marrow neutrophils by expressing soluble extracellular domains of G-CSFR, IL-6R, and gp130in local tissues to block tumor-derived G-CSF and IL-6, or expressing G-CSF and/or IL-6in vivo in absence of tumor. We recruited neutrophils to the tissues at inoculation site by expressing CXCL1in H22hepatocarcinoma model to investigate whether the function of neutrophils could be maintained in blood and in the process of chemotaxis. We recruited neutrophils to peritoneal cavity of mice by using CXCL1-expressing hepatocytes, and isolated the neutrophils from peritoneal cavity for further proof.Results:The neutrophils from bone marrow of naive mice (N-BM) suppressed tumor growth, whereas the neutrophils from bone marrow of tumor-bearing mice (T-BM) promoted tumor growth. G-CSF and IL-6cooperated to modulate bone marrow neutrophils, converting their function from tumor suppressive to tumor promotional. The recruitment of neutrophils suppressed the growth of tumor in control mice, but promoted tumor growth in pG/pI6-mice, suggesting that the function of neutrophils was maintained in blood and in the process of chemotaxis. In co-inoculation test, the neutrophils from peritoneal cavity of naive mice (N-PC) suppressed tumor growth, whereas the neutrophils from that of pG/pI6-mice (pG/pI6-PC) and tumor-bearing mice (T-PC) promoted tumor growth.Conclusion:The above results showed that G-CSF and IL-6modulated the function of neutrophils in bone marrow, and that the function of neutrophils was maintained in blood and in the process of chemotaxis. Objective:To further prove that pre-modulation by G-CSF and IL-6altered the function of neutrophils, we analysis the function of neutrophils in tumor envirenment.Methods:To further prove that pre-modulation by G-CSF and IL-6altered the function of neutrophils, we analyzed the neutrophils recruited to the tissues at inoculation sites.The function of these neutrophils might represent the final function of the neutrophils in tumor milieu, since these neutrophils not only finished the process of chemotaxis, but also were stimulated by complex stimuli in the tissue. To compare the effect of G-CSF/IL-6with that of tumor on neutrophils, we used another model in which the mice received secondary inoculation with tumor cells after the removal of primary tumor. The effect of primary tumor on neutrophils was analyzed by recruiting neutrophils to the tissues at the sites of secondary inoculation.Results:Compared with the neutrophils from control mice, the neutrophils from pG/pI6-mice expressed higher levels of Mmp9and Bv8mRNAs, but lower level of Trail mRNA. The mRNA of Mpo or NE was undetectable or unchanged respectively in the neutrophils isolated from the tissues (data not shown). The neutrophils from pG/pI6-mice contained more intracellular MPO and NE, but much less intracellular TRAIL. We then detected the extracellular TRAIL (soluble TRAIL), MPO, and NE in the tissues. Extracellular TRAIL was detectable in the tissues of control mice, but not pG/pI6-mice. The activities of MPO and NE in local tissues of pG/pI6-mice were significantly lower than those in control mice. For the secondary inoculation model, the results showed that the modulatory effect of G-CSF and IL-6on neutrophils was similar to that of primary tumor. In the test of the function of neutrophils isolated from peritoneal cavity of mice, the neutrophils from N-PC had cytotoxicity to tumor cells in absence or presence of the mixture of soluble molecules from tumor (T-sMs), which might represent complex stimuli in tumor milieu. The cytotoxicity of the neutrophils from pG/pI6-PC and T-PC was significantly lower than that of the neutrophils from N-PC.Conclusion:These data suggested that G-CSF and IL-6could alter the expressions of Trail gene in neutrophils, and attenuate neutrophil degranulation to decrease the release of MPO and NE. The function of the neutrophils was indeed altered by G-CSF and IL-6. Objective:To explore the mechanism involved in the attenuation of neutrophil degranulation, we next analyzed the activation of PI3K and p38MAPK pathways in neutrophils. We further investigated neutrophil degranulation-related protein Rab27a and Rac2. Methods:For this purpose, we stimulated the neutrophils from peritoneal cavity with T-sMs. We analyzed the expressions of Rac2and Rab27a using real-time PCR and Western Blot. WeResults:T-sMs could activate PI3K and p38MAPK pathways in neutrophils. The phosphorylation levels of Akt and p38MAPK in neutrophils from pG/pI6-PC and T-PC were significantly lower than those in neutrophils from N-PC. In vivo expression of G-CSF, but not IL-6, resulted in the inefficient activation of PI3K and p38MAPK pathways. Consistently, in vivo expression of G-CSF attenuated T-sMs-induced degranulation of neutrophils, evaluated by the release of MPO. However, both spontaneous degranulation and induced degranulation of neutrophils from pG/pI6-mice were significantly decreased. In pG/pI6-mice and tumor-bearing mice, the expression of Rab27a gene was decreased in bone marrow neutrophils and the neutrophils recruited to peritoneal cavity. Consistently, the expression of Rab2la gene in neutrophils from N-BM could be down-regulated by co-stimulation with G-CSF and IL-6in vitro. The effect of G-CSF and IL-6was abolished by inhibiting STAT3, but enhanced by inhibiting PI3K. In vivo expression of IFN-P in pG/pI6-mice and tumor-bearing mice increased Rab27a expression in bone marrow neutrophils and the neutrophils recruited to peritoneal cavity. When neutrophils from peritoneal cavity were stimulated with T-sMs, both spontaneous degranulation and induced degranulation of neutrophils from pG/pI6-mice and tumor-bearing mice were significantly decreased, In vivo expression of IFN-β recovered spontaneous degranulation of neutrophils, but failed to fully recover the induced degranulation, since the activation of PI3K and p38MAPK pathways was still inefficient (data not shown). Moreover, IFN-β abrogated tumor-promoting potential of bone marrow neutrophils.Conclusion:These data suggested that the attenuated degranulation was partially attributed to the inefficient activation of PI3K and p38MAPK pathways, and the augmented STAT3activation in neutrophils was another reason for attenuated neutrophil degranulation.