Interleukin-15 (IL-15) is a pro-in£ammatory cytokine known as a general inhibitor of apoptosis, which possesses potential therapeutic properties. Although IL-15 was previously found to be a human neutrophil agonist, its mode of action remains unknown. Herein, we were interested in elucidating the mechanisms by which it delays neutrophil apoptosis. IL-15 was found to induce tyrosine phosphorylation events and to prevent loss of the anti-apoptotic Mcl-1 protein expression. Using di¡erent signal transduction inhibitors, we found that Janus kinase (Jak)-2, Jak-3, p38 mitogen-activated protein kinase (MAPK) and extracellular signal-regulated kinase (ERK), but not G proteins, are involved in IL-15-induced suppression of apoptosis. Furthermore, we found that IL-15 activates Jak-2, p38 MAPK and ERK-1/2, but, unlike granulocyte macrophagecolony-stimulating factor (GM-CSF), it does not activate signal transducer and activator of transcription (STAT)-5a/b. We conclude that IL-15 delays neutrophil apoptosis via several pathways, and that Mcl-1 and several kinases contribute to this. We also conclude that, unlike GM-CSF, IL-15 does not activate the Jak-2/STAT-5 pathway found to be important in neutrophil signaling.

Interleukin-4 (IL-4) is a CD132-dependent cytokine known to activate the Jak-STAT pathway in different cells and cell lines. Although IL-4 has been demonstrated previously to be an agonist in human neutrophils, its capacity to activate different cell signaling pathways in these cells has never been investigated. Two types of IL-4 receptor (IL-4R) exist: the Type I (CD132/IL-4Ralpha heterodimer) and the Type II (IL-4Ralpha/IL-13Ralpha1 heterodimer). In a previous study, we demonstrated that neutrophils express the Type I receptor. Herein, using flow cytometry, we demonstrated that neutrophils, unlike U-937 cells, do not express IL-13Ralpha1 and IL-13Ralpha2 and confirmed the expression of CD132 and IL-4Ralpha on their surface. We also demonstrated that IL-4 induced phosphorylation of Syk, p38, Erk-1/2, JNK, Jak-1, Jak-2, STAT6, and STAT1 and that treatment of cells with the inhibitors piceatannol, SB203580, PD98059, or AG490 reversed the ability of IL-4 to delay neutrophil apoptosis. Using RT-PCR, we demonstrated for the first time that neutrophils express mRNA for all suppressor of cytokine signaling (SOCS) members, namely SOCS1-7 and cytokine-inducible Src homology 2 protein. It is interesting that IL-4 increased expression of SOCS3 at the mRNA and protein levels. The effect of IL-4 on SOCS3 protein expression was increased markedly when the proteasome inhibitor MG132 was added to the cultures, but this was inhibited by cycloheximide, suggesting that SOCS3 is de novo-synthesized in response to IL-4. We conclude that neutrophils express only the Type I IL-4R on their surface and that IL-4 signals via different cell signaling pathways, including the Jak/STAT/SOCS pathway.

Interleukin-15 (IL-15) is a cytokine that possesses interesting, potential therapeutic properties. However, based on several parameters including activation of neutrophils, it is also recognized as a proinflammatory cytokine. The mechanisms by which IL-15 activates human neutrophil functions are not fully understood. Although these cells express a functional IL-15 receptor (IL-15R) composed of IL-15Ralpha, IL-2/15Rbeta (CD122), and gamma(c) (CD132) subunits, the role of each receptor component has not been investigated in IL-15-induced human neutrophil responses. In the present study, fluorescein-activated cell sorter analysis revealed that the ability of IL-15 to enhance neutrophil phagocytosis is not a result of increased expression of IL-15Ralpha, CD122, or CD132 on the neutrophil cell surface. Pretreatment of neutrophils with specific antibodies to IL-15Ralpha, CD122, or CD132 was found to inhibit phagocytosis of opsonized-sheep red blood cells by nearly 40%, 21%, and 27%, respectively. As expected, pretreatment of neutrophils with anti-IL-2Ralpha (CD25) had no effect. Pretreatment of cells with the Syk inhibitor piceatannol was found to significantly inhibit the ability of IL-15 to enhance phagocytosis. In addition, IL-15 was found to induce tyrosine phosphorylation of Syk that was largely inhibited by pretreating cells with piceatannol. Moreover, we found that Syk kinase is physically associated with IL-15Ralpha. We conclude that IL-15R enhances neutrophil phagocytosis by a Syk-dependent mechanism and that the IL-15Ralpha chain plays a key role in mediating this response, at least by interacting with Syk kinase.

Interleukin (IL)-4 is a cytokine known mainly for its anti-inflammatory activity. Using the in vivo murine air pouch model, we found that IL-4 significantly increased the number of leukocytes after 9 hours of treatment, consisting mainly of neutrophil (60%) and monocytic (40%) cell populations. Using an antibody array, we found that the expression of several analytes (predominantly CCL2) was increased by IL-4 before the arrival of leukocytes. The IL-4-induced expression of CCL-2 was confirmed by ELISA. Air pouch resident lining cells were harvested and were found to express IL-4Rα. CCL2 mRNA expression was monitored in lining cells, cells isolated from the air pouch skin, in RAW264.7 macrophage and in epithelial Mode-K cells and its expression was increased in response to IL-4 in all conditions. We conclude that IL-4 can attract leukocytes in vivo by an indirect mechanism involving the production of several analytes by, at least, resident cells.

SUMMARY Viscum album agglutinin‐I (VAA‐I) is a plant lectin which possesses antitumoral properties. This lectin is also known for its immunostimulatory effects when used at low concentrations (1–100 ng/ml). We have demonstrated recently that VAA‐I is a potent inducer of human neutrophil apoptosis in vitro when used at higher concentrations. The role of VAA‐I on activated neutrophils has not so far been investigated and its potential proinflammatory properties in vivo are poorly documented. Herein, we demonstrated that VAA‐I (1000 ng/ml) induces apoptosis in lipopolysaccharide (LPS)‐treated human neutrophils in vitro as well as in murine neutrophils isolated from lipopolysaccharide (LPS)‐induced neutrophil influx. Using this model, we found that administration of VAA‐I (100 or 1000 ng/ml) did not induce an inflammatory response. However, when used at 1 or 10 ng/ml, VAA‐I was found to significantly induce a transitory inflammatory response, based on an increased leucocyte infiltration (>98% neutrophils). Also, we found that VAA‐I inhibits LPS‐induced neutrophil influx when administered simultaneously with LPS. In such conditions, some characteristic apoptotic neutrophils were observed in the pouch. Unlike LPS, which increased the production of some cytokines, VAA‐I (1 or 10 ng/ml) did not increase the production of tumour necrosis factor (TNF)‐α, interleukin (IL)‐1Ra, IL‐1α, IL‐β, IL‐8, IL‐10 or IL‐12 (p70) in human neutrophils. We conclude that VAA‐I possesses the ability to induce apoptosis of preactivated neutrophils at a concentration that does not induce a proinflammatory response. Moreover, we conclude that VAA‐I can inhibit a LPS‐induced proinflammatory response in vivo. These data may provide new clinical perspectives in future mistletoe therapy and favour its potential utilization based on anti‐inflammatory activity that at first appears contradictory with its use as immunostimulant.