Granulocyte/macrophage colony-stimulating factor stimulates the expression of the 5-lipoxygenase-activating protein (FLAP) in human neutrophils.

The synthesis of leukotrienes in human blood neutrophils chiefly relies on the activity of two enzymes, phospholipase A2 and 5-lipoxygenase (5-LO). In turn, the activation of the 5-LO requires the participation of a recently characterized membrane-bound protein, the 5-LO-activating protein (FLAP). In this study, we have investigated conditions under which FLAP expression in neutrophils may be modulated. Of several cytokines tested, only granulocyte/macrophage colony-stimulating factor (GM-CSF) (and to a lesser extent tumor necrosis factor alpha) significantly increased expression of FLAP. GM-CSF increased FLAP mRNA steady-state levels in a time- and dose-dependent manner. The stimulatory effect of GM-CSF on FLAP mRNA was inhibited by prior treatment of the cells with the transcription inhibitor, actinomycin D, and pretreatment of the cells with the protein synthesis inhibitor, cycloheximide, failed to prevent the increase in FLAP mRNA induced by GM-CSF. The accumulation of newly synthesized FLAP, as determined by immunoprecipitation after incorporation of 35S-labeled amino acids, was also increased after incubation of neutrophils with GM-CSF. In addition, the total level of FLAP protein was increased in GM-CSF-treated neutrophils, as determined by two-dimensional gel electrophoresis, followed by Western blot. GM-CSF did not alter the stability of the FLAP protein, indicating that the effect of GM-CSF on FLAP accumulation was the consequence of increased de novo synthesis as opposed to decreased degradation of FLAP. Finally, incubation of neutrophils with the synthetic glucocorticoid dexamethasone directly stimulated the upregulation of FLAP mRNA and protein, and enhanced the effect of GM-CSF. Taken together, these data demonstrate that FLAP expression may be upmodulated after appropriate stimulation of neutrophils. The increase in FLAP expression induced by GM-CSF in inflammatory conditions could confer upon neutrophils a prolonged capacity to synthesize leukotrienes.

Selective activation and functional significance of p38alpha mitogen-activated protein kinase in lipopolysaccharide-stimulated neutrophils.

Activation of leukocytes by proinflammatory stimuli selectively initiates intracellular signal transduction via sequential phosphorylation of kinases. Lipopolysaccharide (LPS) stimulation of human neutrophils is known to result in activation of p38 mitogen-activated protein kinase (MAPk); however, the upstream activator(s) of p38 MAPk is unknown, and consequences of p38 MAPk activation remain largely undefined. We investigated the MAPk kinase (MKK) that activates p38 MAPk in response to LPS, the p38 MAPk isoforms that are activated as part of this pathway, and the functional responses affected by p38 MAPk activation. Although MKK3, MKK4, and MKK6 all activated p38 MAPk in experimental models, only MKK3 was found to activate recombinant p38 MAPk in LPS-treated neutrophils. Of p38 MAPk isoforms studied, only p38alpha and p38delta were detected in neutrophils. LPS stimulation selectively activated p38alpha. Specific inhibitors of p38alpha MAPk blocked LPS-induced adhesion, nuclear factor-kappa B (NF-kappaB) activation, and synthesis of tumor necrosis factor-alpha (TNF-alpha). Inhibition of p38alpha MAPk resulted in a transient decrease in TNF-alpha mRNA accumulation but persistent loss of TNF-alpha synthesis. These findings support a pathway by which LPS stimulation of neutrophils results in activation of MKK3, which in turn activates p38alpha MAPk, ultimately regulating adhesion, NF-kappaB activation, enhanced gene expression of TNF-alpha, and regulation of TNF-alpha synthesis.

Activation of transcription factor NF-kappa B by phagocytic stimuli in human neutrophils.

Phagocytosis represents an important physiological trigger for the inducible expression of several genes in human neutrophils. Here, we report that a DNA-binding activity primarily consisting of the classical NF-kappa B heterodimer, p50/RelA, is induced in phagocytosing neutrophils. Under these conditions, NF-kappa B activation was found to be a rapid and transient response, reaching a maximum by 10-15 min, and returning to near-basal levels by 30 min. In neutrophils undergoing the phagocytosis of opsonized yeasts, the onset of NF-kappa B activation was paralleled by a decline in immunoreactive I kappa B-alpha protein levels, and the cellular I kappa B-alpha pool was replenished by 30 min, in agreement with our gel shift data. We conclude that NF-kappa B activation could constitute one of the mechanisms whereby the expression of kappa B-responsive genes is enhanced in phagocytosing neutrophils. To our knowledge, this represents the first demonstration that phagocytic stimuli can induce NF-kappa B activation in human neutrophils.

Activation of nuclear factor-kappa B by beta-amyloid peptides and interferon-gamma in murine microglia.

An increasing body of evidence suggests that amyloid-beta (A beta) peptides and microglia are crucially involved in the pathogenesis of Alzheimer's disease. In an effort to further elucidate the biological effects of A beta towards microglia, we investigated the ability of A beta peptides to activate nuclear factor (NF)-kappa B in the N9 murine microglial cell line. Co-stimulation of microglia with suboptimal concentrations of A beta(25-35) and 100 U/ml IFN gamma resulted in the detection of a specific NF-kappa B DNA-binding activity in nuclear extracts, as determined in gel mobility shift assays. This response required at least 120 min to be evident and supershift experiments revealed that the NF-kappa B complex contains both RelA and p50. Accordingly, immunoblot experiments showed that amongst NF-kappa B/Rel proteins, RelA and p50 are mobilized to the nucleus following microglial cell stimulation with A beta(25-35) plus IFN gamma. Higher concentrations of A beta(25-35) were effective by themselves in inducing NF-kappa B activation, both in the N9 microglial cell line and in rat primary microglia, as well as in human monocytes. For purposes of comparison, microglia were also stimulated with bacterial LPS, a known NF-kappa B inducer. As expected, LPS strongly induced the formation of two NF-kappa B DNA-binding activities, one of which was identified as RelA/p50. The LPS response was also more rapid, as it was already evident by 40 min and remained sustained for up to 3 h. Collectively, these findings indicate that NF-kappa B activation might constitute one of the mechanisms underlying the inducible expression of kappa B-dependent genes in microglia stimulated by A beta peptides and IFN gamma, or by LPS.

Autocrine enhancement of leukotriene synthesis by endogenous leukotriene B4 and platelet-activating factor in human neutrophils.

1. Platelet-activating factor (PAF) and leukotriene B4 (LTB4), two potent lipid mediators synthesized by activated neutrophils, are known to stimulate several neutrophil functional responses. In this study, we have determined that endogenous LTB4 and PAF exert autocrine effects on LT synthesis, as well as the underlying mechanism involved. 2. Pretreatment of neutrophils with either pertussis toxin (PT), or with receptor antagonists for LTB4 and PAF, resulted in an inhibition of LT synthesis induced by calcium ionophore, A23187. This inhibition was most marked at submaximal (100-300 nM) A23187 concentrations, whilst it was least at ionophore concentrations which induce maximal LT synthesis (1-3 microM). Thus newly-synthesized PAF and LTB4 can enhance LT synthesis induced by A23187 under conditions where the LT-generating system is not fully activated. 3. In recombinant human (rh) granulocyte-macrophage colony-stimulating factor (GM-CSF)-primed neutrophils, LT synthesis in response to chemoattractants (fMet-Leu-Phe or rhC5a) was also significantly inhibited by the LTB4 receptor antagonist, and to a lesser extent by PAF receptor antagonists. 4. Further investigation revealed that LTB4 and/or PAF exert their effects on LT synthesis via an effect on arachidonic acid (AA) availability, as opposed to 5-lipoxygenase (5-LO) activation. Indeed, the receptor antagonists, as well as PT, inhibited LT synthesis and AA release to a similar extent, whereas 5-LO activation (assessed with an exogenous 5-LO substrate) was virtually unaffected under the same conditions. Accordingly, we showed that addition of exogenous LTB4 could enhance AA availability in response to chemoattractant challenge in rhGM-CSF-primed cells, without significantly affecting the 5-LO activation status. Our data show that newly-generated PAF and LTB4 have the ability to positively feedback on LT synthesis by acting at the level of the phospholipase A2/re-esterification component of the LT biosynthetic pathway in neutrophils. Such autocrine affects are likely to represent an important amplification step of LT synthesis, and may as such contribute to the rapid onset, as well as to the evolution, of inflammatory responses.

Activation of the human neutrophil 5-lipoxygenase by leukotriene B4.

1. In the present study, we demonstrate that leukotriene B4 (LTB4) has the ability to activate the human neutrophil 5-lipoxygenase (5-LO). 2. Stimulation of neutrophils with 30 nM 14,15-dideuterio-LTB4 (D2-LTB4) failed to induce the synthesis of LTB4 from endogenous arachidonic acid (AA), but stimulated the formation of LTB4 from 3.3 microM exogenous AA, as determined by GC-MS analysis. 3. The stimulatory effect of LTB4 on 5-LO activity was further examined with an alternative substrate; LTB4 time- and dose-dependently stimulated the 5-LO-mediated conversion of exogenous 15(S)-hydroperoxy-5,8,11,13-(Z,Z,Z,E)-eicosatetraenoate (15-HpETE) into 5(S),15(S)-dihydroxy-6,8,11,13,-(E,Z,Z,E)-eicosatetraenoate (5,15-DiHETE), with a threshold effect at 300 pM. 4. The ability of LTB4 to activate the 5-LO showed structural specificity, since LTB4 was found to be 100 times more potent than omega-hydroxy-LTB4, and 300 times more potent than its delta 6-trans-12-epi-isomer. 5. The LTB4-induced 5-LO activation was effectively inhibited by MK-886 (an inhibitor of 5-LO translocation), by pertussis toxin, and by the LTB4 receptor antagonist, LY-223982. 6. These results demonstrate that the binding of LTB4 to its cell-surface receptor results in 5-LO activation in a process mediated by pertussis toxin-sensitive guanine nucleotide-binding proteins. Our data also suggest that the underlying mechanism involves a translocation of the 5-LO to the membrane. These findings raise the possibility that LTB4 produced by phagocytes may positively feedback on its own synthesis.

Lipopolysaccharide primes neutrophils for a rapid response to IL-10.

Responsiveness of human neutrophils to IL-10 was recently shown to be strictly dependent on the levels of IL-10R1 expression. Activation of signal transducer and activator of transcription 3 (STAT3) phosphorylation and induction of suppressor of cytokine signaling (SOCS)-3 protein by IL-10 are in fact negligible in circulating or freshly isolated ("time 0") neutrophils, but become readily measurable in neutrophils cultured for 4 h in the presence or absence of LPS. In this study, we show that modulation by IL-10 of LPS-induced TNF-alpha, CXCL8/IL-8 and IL-1 receptor antagonist (IL-1ra) mRNA accumulation in neutrophils already expressing a functional IL-10R and antigenic SOCS-3 (i.e. in "4-h-cultured" neutrophils) occurs with kinetics that are similar to those observed in "time 0" neutrophils, depends on de novo protein synthesis, but does not require SOCS-1, SOCS-3, heme oxygenase and Bcl-3 induction. By contrast, we show that IL-10 alone rapidly modulates the expression of TNF-alpha, CXCL8/IL-8 and IL-1ra mRNA, without any new protein synthesis requirement, if neutrophils have been previously exposed to LPS for at least 4 h. These findings suggest that LPS prepares neutrophils to optimally respond to IL-10 in terms of rapid gene modulation via mechanisms that, presumably, depend on specific LPS-induced protein(s).

Interleukin-15 and its impact on neutrophil function.

Interleukin-15 is a recently discovered cytokine produced by several cell types (including fibroblasts, keratinocytes, endothelial cells, and macrophages) in response to endotoxin or microbial infection. In turn, interleukin-15 has been shown to act on various cells of the immune system, including T and B lymphocytes, natural killer cells, monocytes, eosinophils, and circulating neutrophils. In the latter instance, interleukin-15 was initially observed to induce cytoskeletal rearrangements, to enhance phagocytosis, to increase the synthesis of several cellular proteins, and to delay apoptosis. Recently, interleukin-15 has been found to elicit other functional responses in neutrophils, such as chemokine production. This review recapitulates advances made in the area of interleukin-15/neutrophil interactions.

Activation of the NF-kappaB pathway by inflammatory stimuli in human neutrophils.

Activated neutrophils have the ability to upregulate the expression of many genes, in particular those encoding cytokines and chemokines, and to subsequently release the corresponding proteins. Although little is known to date concerning the regulation of gene transcription in neutrophils, it is noteworthy that many of these genes depend on the activation of transcription factors, such as NF-kappaB, for inducible expression. We therefore investigated whether NF-kappaB/Rel proteins are expressed in human neutrophils, as well as their fate on cell activation. We now report that dimers consisting of p50 NFkappaB1, p65 RelA, and/or c-Rel are present in neutrophils and that the greater part of these protein complexes is physically associated with cytoplasmic IkappaB-alpha in resting cells. Following neutrophil stimulation with proinflammatory agonists (such as lipopolysaccharide [LPS], tumor necrosis factor-alpha [TNF-alpha], and fMet-Leu-Phe) that induce the production of cytokines and chemokines in these cells, NF-kappaB/Rel proteins translocated to nuclear fractions, resulting in a transient induction of NF-kappaB DNA binding activity, as determined in gel mobility shift assays. The onset of both processes was found to be closely paralleled by, and dependent on, IkappaB-alpha degradation. Proinflammatory neutrophil stimuli also promoted the accumulation of IkappaB-alpha mRNA transcripts, resulting in the reexpression of the IkappaB-alpha protein. To our knowledge, this constitutes the first indication that NF-kappaB activation may underlie the action of proinflammatory stimuli towards human neutrophil gene expression and, as such, adds a new facet to our understanding of neutrophil biology.

Enhancement by GM-CSF of agonist-induced 5-lipoxygenase activation in human neutrophils involves protein synthesis and gene transcription.

We investigated the effects of granulocyte-macrophage colony-stimulating factor (GM-CSF) on the 5-lipoxygenase (5-LO) component of the leukotriene (LT) biosynthetic pathway of human neutrophils, in order to better understand the mechanism whereby the cytokine primes for LT synthesis. We found that GM-CSF increased 5-LO activation elicited by platelet-activating factor (PAF), N-formyl-methionyl-leucyl-phenylalanine (fMLP), C5a, LTB4, IL-8 and calcium ionophore A23187, as determined by using an exogenous substrate. A close correlation was observed between the priming kinetics of GM-CSF on 5-LO activation and on LT synthesis; moreover, the effects of the cytokine on both 5-LO activation and LT synthesis were inhibited when the cells had been exposed to either the protein synthesis inhibitor, cycloheximide (CX), or the transcription inhibitor, actinomycin D (AD), prior to incubation with GM-CSF. These results raise the possibility that the priming by GM-CSF of LT synthesis may involve an effect of the cytokine on 5-LO protein synthesis and gene expression.

Activation of distinct transcription factors in neutrophils by bacterial LPS, interferon-gamma, and GM-CSF and the necessity to overcome the action of endogenous proteases.

Human neutrophils can be induced to actively transcribe a number of early-response genes, in particular those encoding cytokines, chemokines, and the high-affinity surface receptor for IgG, FcgammaRI. Although little is known to date about the regulation of gene transcription in neutrophils, several indications point to a role for distinct transcription factors, such as members of the NF-kappaB and STAT families. In this study, we investigated whether these transcription factors become activated under stimulatory conditions which are known to induce gene transcription in neutrophils. Unexpectedly, we found that conventional procedures employed to prepare cellular extracts cause the release of proteolytic activities that are normally stored in intracellular granules, resulting in the degradation of various NF-kappaB/Rel and STAT proteins. To circumvent this problem, we developed an alternative procedure which allowed us to show that in neutrophils, LPS and TNFalpha induce a NF-kappaB DNA-binding activity which essentially consists of p50/RelA dimers, and that IFNgamma promotes the binding of STAT1 homodimers to the IFNgamma response region of the FcgammaRI promoter. Moreover, we report that neutrophil stimulation with GM-CSF results in the formation of a STAT5-containing DNA-binding activity. Collectively, the current findings open new perspectives about mechanisms that are likely to regulate gene transcription in neutrophils. In addition, the procedure described herein could prove useful in other cell types that express high levels of endogenous proteases.

Induction by chemokines of lipid mediator synthesis in granulocyte-macrophage colony-stimulating factor-treated human neutrophils.

In recent years, there has been a growing body of evidence suggesting that IL-8 and granulocyte-macrophage CSF (GM-CSF) play an important role in inflammatory processes. We show that after GM-CSF treatment, the exposure of human neutrophils to IL-8 results in the synthesis of leukotriene (LT)B4 and platelet-activating factor. In GM-CSF-treated cells, IL-8 induced a concentration-dependent synthesis of both lipid mediators, with a threshold at 10 to 30 nM, suggesting that IL-8 could stimulate phospholipase A2 activity, an enzyme essential for both syntheses. Accordingly, IL-8 induced a substantial release of 3H-arachidonic acid in GM-CSF-treated PMN. It was also found that IL-8 activates the neutrophil 5-lipoxygenase (5-LO), the other key enzyme in LT biosynthesis. IL-8 induced 5-LO activation in a time- and concentration-dependent manner, with a threshold at 1 nM, and prior treatment of neutrophils with GM-CSF enhanced this effect of IL-8 over the 1 to 300 nM range. Neutrophil-activating peptide-2 and the melanoma growth-stimulatory activity, two peptides that are closely related to IL-8, also had the ability to activate the 5-LO and stimulate LT synthesis, albeit less potently than IL-8. Finally, pertussis toxin and the 5-LO translocation inhibitor, MK-886, both blocked the IL-8-elicited 5-LO activation. Taken together, our results raise the possibility that the combined presence of IL-8 and of GM-CSF at inflammatory foci could result in the synthesis of platelet-activating factor and LTB4 by neutrophils, thereby contributing to the amplification of the inflammatory response.

Transcriptional and translational regulation of inflammatory mediator production by endogenous TGF-beta in macrophages that have ingested apoptotic cells.

We recently reported that phagocytosis of apoptotic cells inhibits the release of inflammatory cytokines by human macrophages. In this paper we show that apoptotic cell uptake by mouse J774 macrophages also inhibits the synthesis and secretion of the chemokines, macrophage inflammatory protein-2 (Mip-2), KC, and Mip-1alpha (but not that of monocyte chemoattractant protein-1 (MCP-1)/JE), and increases TGF-beta formation. Anti-TGF-beta neutralizing Abs largely reversed the inhibitory effect of apoptotic cell uptake, and accordingly, exogenous TGF-beta down-regulated the synthesis of the same mediators. Apoptotic cell ingestion or TGF-beta also inhibited Mip-2 and Mip-1alpha gene expression in LPS-treated J774 cells, whereas TNF-alpha mRNA levels were unaffected. Importantly, TGF-beta pretreatment of J774 cells did not significantly alter chemokine and TNF mRNA stability. Finally, we found that apoptotic cell uptake and TGF-beta did not modulate NF-kappaB or AP-1 DNA binding in J774 cells. We conclude that the decreased production of chemokines and TNF resulting from apoptotic cell ingestion is largely mediated by a common event, i.e., feedback inhibition by endogenous TGF-beta, but involves different mechanisms. Whereas TNF-alpha production appears to be translationally down-regulated, the suppression of most chemokines investigated appears to reflect transcriptional inhibition. In a broader context, the impairment of chemokine and TNF generation by apoptotic cell uptake might represent an important mechanism contributing to the resolution of inflammation. An additional consequence could be the selective recruitment of monocytes into inflammatory sites, as MCP-1/JE production by mouse macrophages was unaffected by apoptotic cell uptake, in contrast to other chemokines.

Granulocyte-macrophage colony-stimulating factor enhances 5-lipoxygenase levels in human polymorphonuclear leukocytes.

Stimulation of human polymorphonuclear leukocytes (PMNL) with granulocyte-macrophage CSF (GM-CSF) results in the enhanced expression of several genes, including some coding for cytokines and enzymes. In this study, we investigated the ability of GM-CSF to up-regulate the human neutrophil 5-lipoxygenase (5-LO), a key enzyme in the leukotriene synthetic pathway. GM-CSF induced a dose- and time-dependent de novo synthesis of the 5-LO in PMNL, as determined by immunoprecipitation of 35S-methionine-labeled 5-LO. This up-regulation occurred within 30 min of treatment with GM-CSF and was observed using concentrations of GM-CSF as low as 30 pM. Prior treatment of the cells with the protein synthesis inhibitor cycloheximide abolished this effect of GM-CSF. Western blot analyses demonstrated that levels of 5-LO did not vary over a 6-h period in unstimulated PMNL treated with CX, and that GM-CSF induced a rapid increase in the total cellular level of 5-LO protein; taken together these results indicated a translational effect of GM-CSF on the expression of the 5-LO. However, GM-CSF did not significantly affect the level of 5-LO mRNA in neutrophils, as determined by Northern blot analysis. Furthermore GM-CSF did not alter the stability of 5-LO mRNA, in agreement with a posttranscriptional effect of GM-CSF on 5-LO expression in PMNL. These results show that human PMNL are capable of up-regulating the expression of the 5-LO in response to physiologic activation.

Lipopolysaccharide-induced interleukin-8 gene expression in human granulocytes: transcriptional inhibition by interferon-gamma.

We recently showed that lipopolysaccharide (LPS) is a potent inducer of interleukin-8 (IL-8) expression in human polymorphonuclear leucocytes (PMN), at the level of both mRNA and protein, and that interferon-gamma (IFN gamma) inhibits IL-8 mRNA accumulation in stimulated PMN. To further define the molecular basis of the regulation of IL-8 gene expression in PMN, we investigated the effects of LPS and IFN gamma at both the transcriptional and post-transcriptional levels. As determined by Northern blot analysis, new protein synthesis was not required for the induction of IL-8 mRNA expression by LPS. Neither did the half-life of IL-8 mRNA in LPS-treated PMN differ from that observed in untreated cells. However, nuclear run-on analysis revealed that LPS increased the transcription of the IL-8 and IL-1 beta genes and that, in LPS-activated cells, IFN gamma markedly inhibited the rate of IL-8 gene transcription, but not that of IL-1 beta. IFN gamma did not affect IL-8 mRNA stability in LPS-treated PMN, indicating that the cytokine does not regulate LPS-induced IL-8 gene expression through post-transcriptional events. These results provide the first evidence that human granulocytes can actively transcribe the IL-8 gene, and that transcriptional inhibition is the mechanism by which IFN gamma inhibits IL-8 gene expression in PMN.

Studies on the activation of human neutrophil 5-lipoxygenase induced by natural agonists and Ca2+ ionophore A23187.

By using exogenous substrates, activation of human neutrophil 5-lipoxygenase can be investigated independently of the release of endogenous arachidonic acid. We have developed a sensitive assay to measure 5-LO activation which takes advantage of the 5-LO-mediated conversion of 15S-hydroperoxy-5,8,11,13(Z,Z,Z,E)-eicosatetraenoic acid (15-HpETE) into 5S,15S-dihydroxy-6,8,11,13(E,Z,Z,E)-eicosatetraenoic acid (5,15-DiHETE). When resting neutrophils were incubated with low micromolar concentrations of 15-HpETE, a minor dose- and time-dependent formation of 5,15-DiHETE was observed. In contrast, co-addition of 15-HpETE with Ca2+ ionophore A23187 or with the neutrophil agonists platelet-activating factor (PAF), fMetLeuPhe or complement component C5a resulted in a sizeable concentration-dependent synthesis of 5,15-DiHETE, while lyso-PAF and phorbol myristate acetate were without effect on 5,15-DiHETE formation from 15-HpETE. This stimulation of 5,15-DiHETE synthesis by A23187 or by natural agonists was effectively inhibited by MK-886, a compound that has recently been reported to inhibit the A23187-induced translocation of 5-LO to membrane structures. Furthermore, natural-agonist-induced activation of the 5-LO-mediated transformation of 15-HpETE was inhibited by pertussis toxin, indicating the involvement of a GTP-binding protein in the 5-LO activation process.

Modulation by interferon-gamma of the production and gene expression of IL-1 receptor antagonist in human neutrophils.

In this report, we show that interferon-gamma (IFN-gamma) modulates the production of IL-1ra in activated human neutrophils. In lipopolysaccharide-stimulated cells, IFN-gamma increased the release of IL-1ra without modulating IL-1ra mRNA accumulation; under these conditions, IFN-gamma only marginally enhanced IL-1ra de novo synthesis, while IL-1ra was more efficiently secreted. In response to the formylated peptide, fMLP, neutrophils released small but significant amounts of IL-1ra, yet without an increase in IL-1ra mRNA over constitutive levels. Following IFN-gamma treatment, however, the fMLP-elicited IL-1ra production was greatly potentiated, and this was accompanied by a transient increased accumulation of IL-1ra mRNA. Finally, opsonized yeast particles were found to induce IL-1ra formation at late incubation times, and prior treatment of neutrophils with IFN-gamma moderately enhanced this response. Collectively, our results demonstrate that in neutrophils activated by different classes of agonists, IFN-gamma modulates the release of IL-1ra by acting through distinct mechanisms.

High affinity receptor for IgG (Fc gamma RI/CD64) gene and STAT protein binding to the IFN-gamma response region (GRR) are regulated differentially in human neutrophils and monocytes by IL-10.

Since IL-10 has been shown to up-regulate the expression of the high affinity receptor for IgG (FcgammaRI/CD64) in human monocytes, we examined whether the cytokine exerts a similar action toward polymorphonuclear neutrophils (PMN). Unexpectedly, we found that in neutrophils, IL-10 failed to induce either the mRNA accumulation or the surface expression of FcgammaRI. Consistent with these findings, stimulation of PMN with IFN-gamma, but not with IL-10, resulted in the induction of specific DNA-binding activities to the IFN-gamma response region (GRR), a regulatory element located in the FcgammaRI gene promoter, required for transcriptional activation. In electrophoretic mobility shift assays (EMSAs), we confirmed that in PBMC, IL-10 induces the binding to the GRR of both STAT1 and STAT3, two members of the STAT family. In neutrophils, however, these activators did not bind to the GRR in response to IL-10, despite the fact that both STAT1 and STAT3 are expressed in these cells. On the other hand, IFN-gamma was an efficient inducer of STAT1 binding to the GRR in both PMN and PBMC. The lack of inducible GRR-binding activity in IL-10-treated PMN could not be ascribed to a lack of IL-10R, and did not appear to reflect an inhibitory effect of the cytokine. Taken together, our data suggest that IL-10 is unable to induce FcgammaRI gene expression in neutrophils because the intracellular signaling pathway triggered by the cytokine is impaired at the level of, or upstream of, STAT1 and/or STAT3 activation.

Interleukin-15 (IL-15) induces NF-kappaB activation and IL-8 production in human neutrophils.

Interleukin-2 (IL-2) and IL-15 exert similar biological actions, which largely reflect the fact that their receptors share common beta and gamma subunits; in contrast, distinct subunits are required for high-affinity binding of either cytokine to a heterotrimeric receptor complex. Human neutrophils are known to express both the beta and gamma subunits of the IL-2/IL-15 receptor complex, and we now report that they also constitutively express messenger RNA transcripts encoding the IL-15 receptor chain, suggesting that they possess functional, heterotrimeric IL-15 receptors. Accordingly, we show that in neutrophils, IL-15 elicits several functional responses. In particular, neutrophils synthesize and release IL-8 in response to IL-15, but not to IL-2. Moreover, a nuclear factor-kappaB (NF-kappaB) DNA-binding activity was enhanced in nuclear extracts of IL-15-treated neutrophils, which could be supershifted by antibodies to p50 or RelA. Again, no detectable effect of IL-2 was observed on this response. In peripheral blood lymphocytes (PBL), however, both IL-2 and IL-15 were potent inducers of NF-kappaB activation. Conversely, neither IL-15 nor IL-2 elicited the formation of activator protein-1 (AP-1) DNA-binding complexes in neutrophils, even though both cytokines were found to activate these DNA-binding activities in PBL. Collectively, these observations establish neutrophils as a useful cellular model to discriminate between the actions of IL-15 and IL-2. More importantly, this is the first demonstration that IL-15 has the ability to induce NF-kappaB and AP-1 activation, which further emphasizes the potential relevance of this newly discovered cytokine to immune and inflammatory processes.