Phagocytosing neutrophils produce and release high amounts of the neutrophil-activating peptide 1/interleukin 8.

After phagocytosis of yeast opsonized with IgG, neutrophil leukocytes (polymorphonuclear leukocytes [PMN]) expressed high levels of neutrophil-activating peptide 1/interleukin 8 (NAP-1/IL-8) mRNA, which peaked after 3-5 h and were still elevated after 18 h. A similar but quantitatively less prominent effect was obtained with lipopolysaccharide (LPS). After phagocytosis, but not after exposure to LPS, the PMN progressively released considerable amounts of NAP-1/IL-8 into the culture medium (18.6-50 ng/ml in 18 h). The peptide released was biologically active, as indicated by the transient elevation of cytosolic-free calcium in PMN exposed to aliquots of the culture supernatants, and desensitization by prestimulation of the cells with recombinant NAP-1/IL-8. By producing NAP-1/IL-8 at sites where they phagocytose invading microorganisms, PMN could enhance the recruitment of new defense cells.

Analysis of tumor necrosis factor promoter responses to ultraviolet light.

Ultraviolet (UV) light induces the biosynthesis of chloramphenicol acetyltransferase (CAT) in the skin of mice bearing the CATTNF reporter transgene. Moreover, nuclear run-on assays indicate that UV light induces transcription of the TNF gene in RAW 264.7 macrophages. These observations suggest that the TNF gene (and/or its mRNA product) responds to signals elicited by UV light. To identify transcriptional UV response elements within the TNF promoter, and to determine whether a posttranscriptional response might also exist, a series of reporter constructs using a CAT coding sequence attached to various portions of the TNF promoter and 3' untranslated region were devised and transfected into several cultured cell lines. All cells tested were found to be UV responsive, and in NIH 3T3 cells, induction was found to depend upon two general regions of the promoter. The more distal region encompassed nucleotides (nt) -1059 through -451 with respect to the cap site, while the more proximal region spanned nt -403 through -261. A negative element, blocking the UV response, was interposed (nt -451 through -403). As with the response to LPS, the response to UV irradiation appears to involve translational activation in macrophages. However, the UV and LPS signaling pathways have little in common with one another, as indicated by three observations. First, no difference in responsiveness was observed on comparison of TNF gene induction in macrophages derived from C3H/HeN as opposed to C3H/HeJ mice. Second, cell fusion studies showed that while the LPS signaling pathway is extinguished by fusion of RAW 264.7 cells with NIH 3T3 cells, the UV signaling pathway remained intact. Finally, induction did not depend upon the NF-kappa B binding sites that are known to be required for LPS response in macrophages, since mutation of these sites did not impair the UV response.

Triggering of antitumor activity through melanoma-specific transduction of a constitutively active tumor necrosis factor (TNF) R1 chimeric receptor in the absence of TNF-alpha.

Tumor necrosis factor-alpha (TNF-alpha) has been intensively studied because of the specific toxicity of this cytokine toward cells that undergo malignant transformation. However, its proinflammatory and immunoregulatory properties always represented a drawback to the TNF-alpha administration in cancer therapy. In this study, we describe an adenovirus-based strategy in which the tumoricidal activity of TNF-alpha can be selectively triggered to eradicate tumors without administering TNF-alpha. This strategy might allow us to prevent TNF-alpha effects on normal tissues and, therefore, to bypass its systemic toxic effects. We inserted the constitutively active version of the Mr 55,000 TNF receptor, which was shown previously (F. Bazzoni et al., Proc. Natl. Acad. Sci. USA, 92: 5376-5380, 1995) to be capable of killing cells upon expression in the absence of its ligand, into a replication-deficient adenovirus, and under the control of a melanoma-specific promoter/enhancer element. We show that, upon infection, the recombinant gene reaches high level of expression in melanoma cell lines and triggers apoptosis by activating the caspase cascade. Expression and function of this receptor is restricted to melanoma cell lines, because morphology, viability, and proliferation of other cell types exposed to the recombinant adenovirus infection are not affected. We show for the first time that a TNF-like apoptotic response can be triggered in the absence of TNF-alpha and can be selectively confined to specific cellular targets. Killing activity and tissue specificity of the recombinant TNF receptor adenovirus, together with the advantage of triggering a TNF-like antitumor activity in the absence of TNF-alpha itself, are ideal features for a vector that might be the choice for gene therapy aimed to eradicate malignant cells.

Phagocytosis of opsonized yeast induces tumor necrosis factor-alpha mRNA accumulation and protein release by human polymorphonuclear leukocytes.

In this report we show that phagocytosis of yeast particles opsonized with IgG (Y-IgG) by human polymorphonuclear cells (PMN) results in the selective induction of tumor necrosis factor (TNF-alpha) messenger RNA (mRNA) and release of its mature protein. Lipopolysaccharide (LPS) was also found able to induce TNF-alpha secretion by PMN, but was a less potent stimulus compared with Y-IgG. There was no evidence of interleukin-6 (IL-6) gene expression in PMN after phagocytosis of Y-IgG or in response to LPS, whereas IL-6 mRNA expression and secretion were induced by either stimulus in monocytes. These findings demonstrate that a physiological function such as phagocytosis modulates the gene expression for a cytokine in PMN and shed new light on the understanding of the pathogenesis of the inflammatory process.

Amiloride does not influence the capability of interferon gamma to potentiate superoxide anion and hydrogen peroxide release by human mononuclear phagocytes.

Since the molecular mechanisms of macrophage activation in response to interferon gamma (IFN-gamma) are still not well defined we have investigated whether amiloride, a specific inhibitor of the Na+/H+ antiporter, had any effect on the IFN-gamma-mediated potentiation of human monocyte and monocyte-derived macrophage capability to produce O2- (respiratory burst). Here, we demonstrate that amiloride neither inhibits the capability of IFN-gamma to activate the mononuclear phagocyte respiratory burst nor influences IFN-gamma induction of steady-state mRNA levels for 2 components of the superoxide anion-generating enzyme system. On the contrary, we show that IFN-gamma-enhanced expression of the HLA-DR alpha gene is significantly inhibited by amiloride These data indicate that Na+/H+ antiporter stimulation by IFN-gamma is not involved in the mechanism of activation of macrophage oxidative metabolism.

Comparative expression of TNF-alpha alleles from normal and autoimmune-prone MHC haplotypes.

The tumor necrosis factor-alpha (TNF-alpha, or TNF) genes of NOD mice and NZW mice are reportedly underexpressed relative to the TNF genes of control mice in lipopolysaccharide (LPS)-induced peritoneal macrophages. These findings, as well as the well-known major histocompatibility complex (MHC) linkage of the TNF genes, have prompted speculation that mutations affecting expression of the TNF-alpha loci might represent a primary cause of autoimmune diseases. Differences in expression of the TNF genes in different strains of mice might result either from effects of cis-acting mutations or from differences in the cellular environment that operate in trans. To discriminate between these possibilities, we directly examined the relative contribution made by each of two different TNF alleles (one associated with an autoimmune-prone haplotype and the other not) to the pool of TNF mRNA within the cells of F1 hybrid mice. Reverse transcription-polymerase chain reaction (RT-PCR) was used to amplify a polymorphic fragment derived from the total pool of TNF mRNA present in LPS-induced peritoneal macrophages of hybrid animals produced by crossing BALB/c to non-obese diabetic (NOD), and New Zealand black (NZB) to New Zealand white (NZW). In both types of F1 hybrid, the two alleles were represented in nearly equal quantities at the mRNA level. It may be inferred that at all pretranslational levels, the NZW and NOD TNF alleles are functionally equivalent to the control alleles that were examined. Interstrain differences in responsiveness to LPS are therefore responsible for interstrain differences in TNF gene expression.

How do tumor necrosis factor receptors work?

Quite suddenly, a new level of understanding has been attached to the TNF ligand and receptor families. Many of the proximal transducers that signal the presence of TNF or its homologs have been identified, and certain components of the distal signaling pathway have emerged as well. We lack, however, a "movie" of the events that transpire when TNF binds its receptors on the surface of a cell. The facts in hand permit an educated approximation.

MIP-1 gamma: molecular cloning, expression, and biological activities of a novel CC chemokine that is constitutively secreted in vivo.

We have identified a novel CC chemokine family member, herein termed MIP-1 gamma in view of its similarity to existing members of the MIP-1 group. The murine protein has a predicted length of 100 amino acids. Like MIP-1 alpha, recombinant MIP-1 gamma acts as a pyrogen when administered intracerebroventricularly. MIP-1 gamma and MIP-1 alpha engage the same high-affinity receptor on neutrophils, activating calcium release within seconds following cell contact. Pretreatment with either chemokine abolishes responses to the other, and to itself, suggesting utilization of a common signaling pathway. However, unlike MIP-1 alpha or any of the other CC chemokines, MIP-1 gamma is expressed constitutively by a wide variety of tissues, and circulates in the blood of healthy mice at concentrations of approximately 1 microgram/ml (90 nM). It would therefore be predicted that MIP-1 gamma occupies most of the CC chemokine receptors that exist in the intravascular compartment. As such it might, under normal circumstances, markedly influence responses to the inducible CC chemokines.

TNF, apoptosis and autoimmunity: a common thread?

A subset of cytokine mediators belonging to the tumor necrosis factor (TNF) family cause apoptosis, acting through receptors and signaling pathways that have recently come to light. Further, at least one autoimmune disease results from a defined defect of apoptosis (mutations of the Fas ligand or its receptor). It is offered that many, and perhaps most autoimmune diseases may result from primary defects of apoptosis. Such defects may cause reflexive overproduction of TNF and other pro-apoptotic cytokines. The collateral damage produced by these mediators may be of pathogenetic importance in complex autoimmune disorders such as rheumatoid arthritis and Crohn disease, wherein TNF blockade is known to have ameliorative effects.

Chimeric tumor necrosis factor receptors with constitutive signaling activity.

Many hormone and cytokine receptors are crosslinked by their specific ligands, and multimerization is an essential step leading to the generation of a signal. In the case of the tumor necrosis factor (TNF) receptors (TNF-Rs), antibody-induced crosslinking is sufficient to trigger a cytolytic effect. However, the quaternary structural requirements for signaling--i.e., the formation of dimers, trimers, or higher-order multimers--have remained obscure. Moreover, it has not been clear whether the 55-kDa or 75-kDa TNF-R is responsible for initiation of cytolysis. We reasoned that an obligate receptor dimer, targeted to the plasma membrane, might continuously signal the presence of TNF despite the actual absence of the ligand. Such a molecule, inserted into an appropriate vector, could be used to project receptor-specific "TNF-like" activity to specific cells and tissues in vivo. Accordingly, we constructed sequences encoding chimeric receptors in which the extracellular domain of the mouse erythropoietin receptor (Epo-R) was fused to the "stem," transmembrane domain, and cytoplasmic domain of the two mouse TNF-Rs. Thus, the Epo-R group was used to drive dimerization of the TNF-R cytoplasmic domain. These chimeric proteins were well expressed in a variety of cell lines and bound erythropoietin at the cell surface. Both the 55-kDa and the 75-kDa Epo/TNF-R chimeras exerted a constitutive cytotoxic effect detected by cotransfection or clonogenic assay. Thus, despite the lack of structural homology between the cytoplasmic domains of the two TNF-Rs, a similar signaling endpoint was observed. Moreover, dimerization (rather than trimerization or higher-order multimerization) was sufficient for elicitation of a biological response.

Studies on molecular regulation of phagocytosis in neutrophils. Con A-mediated ingestion and associated respiratory burst independent of phosphoinositide turnover, rise in [Ca2+]i, and arachidonic acid release.

The role of the activation of phosphoinositide turnover and of the increase in cytosolic free calcium, [Ca2+]i, in the phagocytosis and associated activation of the respiratory burst was investigated. We report the results obtained on the phagocytosis of yeast cells mediated by Con A in normal and in Ca2+-depleted human neutrophils. In normal neutrophils the phagocytosis was associated with a respiratory burst, a stimulation in the formation of [3H] inositol phosphates and [32P]phosphatidic acid, the release of [3H]arachidonic acid, and a rise in [Ca2+]i. Ca2+-depleted neutrophils are able to perform the phagocytosis of yeast cells mediated by Con A and to activate the respiratory burst without stimulation of [3H]inositol phosphates and [32P]phosphatidic acid formation, [3H]arachidonic acid release, and rise in [Ca2+]i. In both normal and Ca2+-depleted neutrophils the phagocytosis and the associated respiratory burst, 1) were inhibited by cytochalasin B; 2) were insensitive to H-7, an inhibitor of protein kinase C; and 3) did not involve GTP-binding protein sensitive to pertussis toxin. These findings indicate that the activation of phosphoinositide turnover, the liberation of arachidonic acid, the rise in [Ca2+]i, and the activity of protein kinase C are not necessarily required for ingestion of Con A-opsonized particles and for associated activation of the NADPH oxidase, the enzyme responsible for the respiratory burst. The molecular mechanisms of these phosphoinositide and Ca2+-independent responses are discussed.

Interferon-gamma transcriptionally modulates the expression of the genes for the high affinity IgG-Fc receptor and the 47-kDa cytosolic component of NADPH oxidase in human polymorphonuclear leukocytes.

We examined the mechanisms responsible for the regulation by interferon-gamma (IFN-gamma) of the expression of the genes encoding the high affinity IgG-Fc receptor (Fc gamma R-I, CD64) and the NADPH oxidase 47-kDa cytosolic factor (p47-phox) in human polymorphonuclear leukocytes (PMN). Nuclear run-on transcriptional assays demonstrated that the Fc gamma R-I gene transcription is undetectable in untreated PMN but is significantly induced by IFN-gamma. Unlike Fc gamma R-I, p47-phox gene transcription is constitutively active in resting PMN and is down-regulated by a 2-h treatment of these cells with IFN-gamma. The transcriptional modulation by IFN-gamma of Fc gamma R-I and p47-phox genes is not influenced by the protein synthesis inhibitor cycloheximide. Moreover, Northern blot analysis revealed that cycloheximide superinduces p47-phox mRNA expression by increasing its half-life and without affecting p47-phox gene transcription. These findings indicate that human PMN can regulate gene expression by transcriptional and posttranscriptional events.

Evidence for the involvement of distinct signal transduction pathways in the regulation of constitutive and interferon gamma-dependent gene expression of NADPH oxidase components (gp91-phox, p47-phox, and p22-phox) and high-affinity receptor for IgG (Fc gamma R-I) in human polymorphonuclear leukocytes.

We recently showed that mRNA levels coding the high-affinity Fc gamma receptor for IgG (Fc gamma R-I, CD64) and two of the components of the phagocytic superoxide anion-generating system--the heavy-chain subunit of cytochrome b558 (gp91-phox) and the 47-Kd cytosolic factor (p47-phox)--are modulated by interferon gamma (IFN-gamma). In this study, we examined whether dexamethasone (DEX) affects gp91-phox and p47-phox mRNA expression of human polymorphonuclear leukocytes (PMN), treated or not with IFN-gamma. We also investigated whether staurosporine, a general inhibitor of protein kinases, influences gp91-phox, p47-phox, and Fc gamma R-I gene expression in PMN treated with or without IFN-gamma. We found that (1) gp91-phox mRNA steady-state levels, expressed in control or IFN-gamma-treated PMN, were significantly inhibited, in a dose-dependent fashion, by both DEX and staurosporine; (2) p47-phox mRNA steady-state levels, expressed in control or IFN-gamma-treated PMN, were not influenced by DEX, but were markedly depressed by staurosporine; (3) no changes of spectrophotometric cytochrome b558 were found in PMN treated for up to 20 hours with the inhibitors, regardless of the presence of IFN-gamma; (4) both DEX and staurosporine dose-dependently inhibited IFN-gamma-induced Fc gamma R-I mRNA and protein expression; and (5) stability of gp91-phox and Fc gamma R-I messages in IFN-gamma-treated PMN was not altered by the presence of DEX. Our results demonstrate that gp91-phox, p47-phox, and Fc gamma R-I gene expression of PMN is governed by specific and independent biochemical pathways. Moreover, IFN-gamma activates different signal transduction pathways to modulate mRNA expression of gp91-phox, p47-phox, and Fc gamma R-I.

IL-8 production by human polymorphonuclear leukocytes. The chemoattractant formyl-methionyl-leucyl-phenylalanine induces the gene expression and release of IL-8 through a pertussis toxin-sensitive pathway.

IL-8 is a novel chemotactic cytokine, produced by a variety of blood and tissue cells, that has marked activating effects on polymorphonuclear leukocytes (PMN). We report that IL-8 is produced and released by human PMN after stimulation with the chemotactic agonist FMLP. Release of IL-8 in response to FMLP was transient and not influenced by PMN adherence or by the absence of serum in the medium. Maximum yields were usually obtained with 10 nM FMLP within 2 h of stimulation (0.5-3.5 ng/ml/7 x 10(6) cells, range of 17 different donors). IL-8 release was dependent on FMLP-induced de novo protein synthesis because it was inhibited by cycloheximide, was paralleled by enhanced expression of IL-8 mRNA and was potentiated from two- to sixfold after preincubation of PMN with cytochalasin B. The FMLP effect was direct and not dependent on LPS or on contaminating monocytes, which showed only low responsiveness to FMLP. Pretreatment of PMN with pertussis toxin prevented FMLP-dependent IL-8 production, the effect being evident both at the level of mRNA expression and protein secretion. In addition, two other chemoattractans, platelet-activating factor and C5a, were found capable to induce release of IL-8 by PMN. The results of this study suggest that chemotactically stimulated PMN may be able to amplify the recruitment process of PMN to the inflammatory site by releasing IL-8. As a long-lived cytokine, IL-8 could markedly prolong the attractant effect.

Interferon-gamma inhibits interleukin-8 production by human polymorphonuclear leucocytes.

Stimulation of human polymorphonuclear leucocytes (PMN) with phagocytosable particles [yeast-IgG (Y-IgG)], lipopolysaccharide (LPS), tumour necrosis factor (TNF) or formyl-methionyl-leucyl-phenyl-alanine (FMLP) results in an increase of the interleukin-8 (IL-8) mRNA accumulation and a subsequent release of the protein. Here, we report that interferon-gamma (IFN-gamma) down-regulates the constitutive IL-8 mRNA levels expressed by resting PMN. As shown by Northern analysis, this down-modulation occurred rapidly, was not dependent on new protein synthesis, and was not caused by an increased rate of degradation of IL-8 mRNA. Preincubation of PMN with IFN-gamma significantly inhibited their ability to release IL-8 upon stimulation with TNF, LPS, FMLP and Y-IgG, but enhanced the respiratory burst capability in response to FMLP and TNF. TNF-, LPS- and FMLP-induced expression of IL-8 mRNA was also selectively inhibited by IFN-gamma. Taken together these findings suggest that IFN-gamma has important regulatory effects on acute inflammatory response because of its capacity to modulate negatively IL-8 gene expression and secretion by human PMN. Further observations revealed that, in human PMN, degradation of IL-8 mRNA is finely regulated, and that cycloheximide (CHX), an inhibitor of protein synthesis, super-induces the mRNA accumulation for IL-8 in a dose- and time-dependent manner.

Activation of human neutrophils by substance P. Effect on oxidative metabolism, exocytosis, cytosolic Ca2+ concentration and inositol phosphate formation.

The neuropeptide substance P (SP), which has been suggested to mediate neurogenic inflammation, induces in human neutrophils the activation of the respiratory burst measured as O2 consumption and H2O2 production, and a cytochalasin B-dependent secretion of specific and azurophilic granules. The SP(4-11) fragment is much more stimulant than the entire molecule, whereas the SP(1-4) fragment is inactive. The respiratory and secretory response to SP are associated with an activation of phosphoinositide turnover, of Ca2+ influx and release from intracellular stores. Pertussis toxin inhibits 70% of the respiratory response and the residual 30% activity remains, even increasing 10-fold the concentration of the toxin. 1-(5-Isoquinolinesulfonyl)-2-methylpiperazine, a putative inhibitor of protein kinase C, does not modify the respiratory response to SP. Cytochalasin B significantly depresses the activation of the respiration by SP, whereas it moderately enhances the activation of phosphoinositide turnover and potentiates the increase of intracellular Ca2+ concentration. The results are discussed in relation to the receptor apparatus involved in SP activity, the signal transduction sequence activated by SP for the stimulation of NADPH oxidase, and the role of cell response to SP in the inflammatory process.

Analysis of the Bak protein expression in human polymorphonuclear neutrophils.

In this study, we investigated the expression of Bak, a member of the Bcl-2 protein family, in human polymorphonuclear neutrophils. Northern blot and Western blot analyses revealed that Bak messenger RNA and protein were constitutively expressed in peripheral polymorphonuclear neutrophils and mononuclear cells, as well as in several hematopoietic cell lines. Remarkably, culturing neutrophils for 24 h in the presence or absence of interferon-gamma or tumor necrosis factor-alpha, which have been described to modulate the survival rate of these cells, did not influence the expression of antigenic Bak. Taken together, our data indicate that the expression of the pro-apoptotic protein Bak in polymorphonuclear neutrophils is constitutive, is not subject to modulation, and does not correlate with the neutrophil life span in culture.

Isolation and characterization of a cDNA clone for a novel serine-rich neutrophil protein.

A cDNA expression library from pig blood neutrophils was immunoscreened with a rabbit antiserum raised against a 32 kDa neutrophil membrane phosphoprotein. Previous work indicated this protein as a component of the superoxide-forming NADPH oxidase enzyme complex (1,2). Only one cDNA clone (B+) was highly positive. The B+ clone contained a 1109 bp insert, with an open reading frame encoding for 284 amino acids. The deduced B+ amino acid sequence contained a 72 amino acid domain with proline and glutamine repeats and two domains extremely enriched with serine residues. The isolated cDNA hybridizes with a 3.1 kb mRNA expressed in pig and human leukocytes.

Molecular basis of interferon-gamma and lipopolysaccharide enhancement of phagocyte respiratory burst capability. Studies on the gene expression of several NADPH oxidase components.

In this study, we analyzed the expression of genes encoding for components of the phagocyte superoxide anion-generating system in human phagocytes treated with interferon-gamma (IFN-gamma) or lipopolysaccharide (LPS). Human neutrophils express high levels of the 47-kDa cytosolic factor (p47-phox), which are down-regulated after treatment with IFN-gamma, but not with LPS. On the contrary, the steady-state levels of the heavy chain subunit of cytochrome b558 (gp91-phox) were increased by IFN-gamma and LPS in human monocyte-derived macrophages and neutrophils in a time- and dose-dependent fashion, whereas cytochrome b558 light chain subunit (p22-phox) mRNA was not influenced by either agent. Studies on post-transcriptional regulation at the level of mRNA stability indicate that, in neutrophils, IFN-gamma has no influence on gp91-phox and p47-phox mRNA half-lives. The content of the two cytochrome b558 subunits was quantified by enzyme-linked immunosorbent assay, which revealed that, in neutrophils, gp91-phox levels doubled after 4 h of treatment with IFN-gamma or LPS. Monocyte/macrophage maturation was associated with a gradual decrease in gp91-phox mRNA and protein levels, which were both restored by treatment with IFN-gamma for 24-48 h. These results suggest that induction of the gp91-phox gene and protein product by IFN-gamma or LPS is an important requirement in the mechanism of the enhancement of neutrophil and macrophage oxidative metabolism.

The neutrophil as a cellular source of chemokines.

Neutrophils are known to play an important role in inflammatory responses by virtue of their ability to perform a series of effector functions that collectively represent a major mechanism of innate immunity against injury and infection. In recent years, however, it has become obvious that the contribution of neutrophils to host defence and natural immunity extends well beyond their traditional role as professional phagocytes. Indeed, neutrophils can be induced to express a number of genes whose products lie at the core of inflammatory and immune responses. These include not only Fc receptors, complement components, cationic antimicrobial and NADPH oxidase proteins, but also a variety of cytokines (including tumour necrosis factor-alpha, interleukin (IL)-1beta, IL-1R alpha, IL-12 and vascular endothelial growth factor), and chemokines such as IL-8, growth-related gene product, macrophage inflammatory protein (MIP)-1alpha, MIP-1beta, interferon-gamma-inducible protein of 10 kDa and monokine induced by interferon-gamma. Because these chemokines are primarily chemotactic for neutrophils, monocytes, immature dendritic cells and T-lymphocyte subsets, a potential role for neutrophils in orchestrating the sequential recruitment of distinct leukocyte types to the inflamed tissue is likely to occur. The purpose of this review is to summarize the essential features of the production of chemokines by polymorphonuclear neutrophil leukocytes and the contribution that we have made to characterize some aspects of this newly discovered crucial function of neutrophils.