Leishmania major and Leishmania donovani: effect of LPG-containing and LPG-deficient strains on monocyte chemotaxis and chemiluminescence.

Lipophosphoglycan (LPG) is a major glycolipid present on the membrane of Leishmania promastigotes and amastigotes. We have previously shown that preincubation of peripheral blood monocytes with purified LPG inhibits IL-1 production, chemotactic locomotion, and luminol-dependent chemiluminescence (LDCL). In the present study we tested the effect of LPG present on live parasites on monocyte activity. For this purpose, we used two mutant strains deficient in LPG and two LPG-containing strains. One pair was Leishmania major and the other Leishmania donovani. Monocytes in suspension were infected with the different parasite strains and tested for chemotactic locomotion and LDCL at different times between 1 and 72 hr after infection. In parallel, the percentage of infected monocytes was measured in stained cytospin preparations. The results obtained showed that at 1 hr of incubation only the LPG-containing strains inhibited chemotaxis, while the mutant strains showed a normal response. From 4 hr of incubation onwards, the mutant strains also inhibited monocyte chemotactic locomotion. LDCL was only slightly inhibited by the LPG-containing strains after 1 hr, because of a high level of spontaneous stimulation, probably due to phagocytosis. At 24 and 72 hr all strains inhibited LDCL. These results suggest that LPG is responsible for early inhibition of macrophage activity, but that other factors are responsible for inhibition at later stages of in vitro infection. The model described here might represent a useful tool to further analyze the mechanisms involved in immune evasion of Leishmania parasites.     

Quantitation of Leishmania lipophosphoglycan repeat units by capillary electrophoresis

The glycosylphosphatidylinositol (GPI)-anchored lipophosphoglycan (LPG) of Leishmania is the dominant cell surface glycoconjugate of these pathogenic parasites. LPG is structurally characterized by a series of phosphoglycan repeat units. Determining the number of repeat units per LPG molecule has proven difficult using current technologies, such as mass spectrometry. As an alternative method to quantitate the number of repeat units in LPG, a procedure based on capillary electrophoretic analysis of the proportion of mannose to 2,5-anhydromannose (derived from the nonacetylated glucosamine of the GPI anchor of LPG) was developed. The CE-based technique is sensitive and relatively rapid compared to GC-MS-based protocols. Its application was demonstrated in quantitating the number of LPG repeat units from several species of Leishmania as well as from two life-cycle stages of these organisms.     

Leishmania major: Reactive oxygen species and interferon gamma induction by soluble lipophosphoglycan of stationary phase promastigotes

Protozoan parasites of the genus Leishmania cause a number of important human diseases. One of the key determinants of parasite infectivity and survival is membrane glycoconjugate lipophosphoglycan (mLPG). In addition, it has been shown that mLPG could be used as a transmission blocking vaccine. Since culture supernatant of parasite promastigotes is a good source of LPG, we attempted to compare the immunological properties of culture supernatant and membrane LPG prepared from stationary phase promastigotes of Leishmania major. The purity of supernatant LPG (sLPG) and membrane LPG (mLPG) was determined by thin layer chromatography. The effect of sLPG and mLPG on the production of reactive oxygen species (ROS) was studied using PBMCs isolated from healthy individuals. In addition, induction of IL-12, IFN-gamma and IL-10 secretion in the presence of sLPG and mLPG was investigated. Reactive oxygen species in addition to IL-10 and IL-12 were induced by both sLPG and mLPG. However, IFN-gamma production was promoted only in response to sLPG suggesting its ability to promote Th1 response and implication in vaccine design.     

IL-4 inhibits H2O2 production and antileishmanial capacity of human cultured monocytes mediated by IFN-gamma

The effect of IL-4 on the IFN-gamma-induced state of activation of cultured human monocytes was investigated with regard to their ability to produce hydrogen peroxide and their antileishmanial capacity towards the intracellular parasite Leishmania donovani. IL-4 was found to inhibit the IFN-gamma-dependent hydrogen peroxide production of monocytes. Treatment of monocytes with IFN-gamma (200 to 600 U/ml) for 48 h increased the hydrogen peroxide production fourfold above background. Coincubation of the monocytes with IL-4 (1 to 1000 U/ml) and IFN-gamma (200 to 600 U/ml) inhibited this increase by 50 to 100%. IL-4 alone did not modulate the hydrogen peroxide production of monocytes. Pretreatment of monocytes with IL-4 for 20 min to 3 h was already effective in preventing the IFN-gamma response. Addition of IL-4 not later than 6 h after the start of incubation with IFN-gamma was necessary for an optimal inhibitory effect. IL-4 also inhibited the IFN-gamma-induced antileishmanial capacity of monocytes: IFN-gamma (1000 U/ml) induced a 54 +/- 10% reduction in the number of parasites. Monocytes treated with combinations of IL-4 (100 to 1000 U/ml) and IFN-gamma (1000 U/ml) were unable to reduce the parasite numbers. IL-4 alone did not alter the uptake of Leishmania donovani nor induce antileishmanial activity. These results demonstrate that IL-4 disables human cultured monocytes to respond to IFN-gamma activation.     

Surface determinants of Leishmania parasites and their role in infectivity in the mammalian host

Leishmania are intracellular protozoan parasites that reside primarily in host mononuclear phagocytes. Infection of host macrophages is initiated by infective promastigote stages and perpetuated by an obligate intracellular amastigote stage. Studies undertaken over the last decade have shown that the composition of the complex surface glycocalyx of these stages (comprising lipophosphoglycan, GPI-anchored glycoproteins, proteophosphoglycans and free GPI glycolipids) changes dramatically as promastigotes differentiate into amastigotes. Marked stage-specific changes also occur in the expression of other plasma membrane components, including type-1, polytopic and peripheral membrane proteins, reflecting the distinct microbicidal responses and nutritional environments encountered by these stages. More recently, a number of Leishmania mutants lacking single or multiple surface components have been generated. While some of these mutants are less virulent than wild type parasites, many of these mutants exhibit only mild or no loss of virulence. These studies suggest that, 1) the major surface glycocalyx components of the promastigote stage (i.e. LPG, GPI-anchored proteins) only have a transient or minor role in macrophage invasion, 2) that there is considerable functional redundancy in the surface glycocalyx and/or loss of some components can be compensated for by the acquisition of equivalent host glycolipids, 3) the expression of specific nutrient transporters is essential for life in the macrophage and 4) the role(s) of some surface components differ markedly in different Leishmania species. These mutants will be useful for identifying other surface or intracellular components that are required for virulence in macrophages.     

Quantitation of Leishmania lipophosphoglycan repeat units by capillary electrophoresis

The glycosylphosphatidylinositol (GPI)-anchored lipophosphoglycan (LPG) of Leishmania is the dominant cell surface glycoconjugate of these pathogenic parasites. LPG is structurally characterized by a series of phosphoglycan repeat units. Determining the number of repeat units per LPG molecule has proven difficult using current technologies, such as mass spectrometry. As an alternative method to quantitate the number of repeat units in LPG, a procedure based on capillary electrophoretic analysis of the proportion of mannose to 2,5-anhydromannose (derived from the nonacetylated glucosamine of the GPI anchor of LPG) was developed. The CE-based technique is sensitive and relatively rapid compared to GC-MS-based protocols. Its application was demonstrated in quantitating the number of LPG repeat units from several species of Leishmania as well as from two life-cycle stages of these organisms.     

Identification of genes encoding arabinosyltransferases (SCA) mediating developmental modifications of lipophosphoglycan required for sand fly transmission of leishmania major

At key steps in the infectious cycle pathogens must adhere to target cells, but at other times detachment is required for transmission. During sand fly infections by the protozoan parasite Leishmania major, binding of replicating promastigotes is mediated by galactosyl side chain (scGal) modifications of phosphoglycan repeats of the major surface adhesin, lipophosphoglycan (LPG). Release is mediated by arabinosyl (Ara) capping of LPG scbetaGal residues upon differentiation to the infective metacyclic stage. We used intraspecific polymorphisms of LPG structure to develop a genetic strategy leading to the identification of two genes (SCA1/2) mediating scAra capping. These LPG side chain beta1,2-arabinosyltransferases (scbetaAraTs) exhibit canonical glycosyltransferase motifs, and their overexpression leads to elevated microsomal scbetaAraT activity. Although the level of scAra caps is maximal in metacyclic parasites, scbetaAraT activity is maximal in log phase cells. Because quantitative immunolocalization studies suggest this is not mediated by sequestration of SCA scbetaAraTs away from the Golgi apparatus during log phase, regulation of activated Ara precursors may control LPG arabinosylation in vivo. The SCA genes define a new family of eukaryotic betaAraTs and represent novel developmentally regulated LPG-modifying activities identified in Leishmania.     

The Leishmania donovani lipophosphoglycan T lymphocyte-reactive component is a tightly associated protein complex

Lymphocytes from mice immunized with Leishmania donovani (LPG) were specifically stimulated to proliferate in vitro by purified LPG or its delipidated congener, phosphoglycan. The response was dose dependent and required prior immunization with either LPG or phosphoglycan. Proliferation was eliminated by specific depletion of Thy-1+ cells with antisera and C and the proliferating T cell subset was shown to be CD4+CD8-. Tests of various LPG fragments indicated that the T cell stimulation was associated with the core structure of LPG rather than the lipid or phosphoglycan repeat structure. However, amino acid analysis of LPG and active LPG fragments, after acid hydrolysis, showed the presence of amino acids in peptide linkage. Specific hydrolysis of the glycosidic linkages in LPG with trifluoromethanesulfonic acid provided polypeptide material reactive with two mAb previously believed to be LPG carbohydrate core specific. The protein was separated from LPG by reverse phase chromatography and shown to be a complex of proteins with common epitopes recognized by the two mAb. The dominant species isolated from LPG was a set of small, approximately 11,000 Mr, molecules. Subsequent T cell proliferation studies showed that the lymphocyte stimulation was associated with the protein component of LPG and not the glycan.     

Identification of the defect in lipophosphoglycan biosynthesis in a non-pathogenic strain of Leishmania major.

The major macromolecule on the surface of the protozoan parasite, Leishmania major, is a complex lipophosphoglycan (LPG), which is anchored to the plasma membrane by an inositol-containing phospholipid. A defect in LPG biosynthesis is thought to be responsible for the avirulence of the L. major strain LRC L119 in mice. In order to identify the nature of this defect we have characterized two truncated forms of LPG, which are accumulated in this strain, by one- and two-dimensional 500-MHz 1H NMR spectroscopy, two-dimensional heteronuclear 1H-31P NMR spectroscopy, methylation analysis, and exoglycosidase digestions. The structures of these glycoinositolphospholipids, termed GIPL-4 and -6, are as follows: [formula: see text] The glycan moieties of GIPL-4 and -6 are identical to the anchor region of LPG, which is also substituted with a Glc-1-PO4 residue in approximately 60% of the structures. However, instead of being capped with chains of phosphorylated oligosaccharide repeat units, both glycan moieties terminate in Man alpha 1-PO4, suggesting that the defect in LPG biosynthesis is in the transfer of galactose to this residue to form the disaccharide backbone of the first repeat unit. These results indicate that the phosphoglycan moiety of LPG is essential for intracellular survival of the parasite and have implications for LPG biosynthesis.     

Structural characterization of a novel class of glycophosphosphingolipids from the protozoan Leptomonas samueli.

Aqueous phenol extraction of the lower trypanosomatid Leptomonas samueli released into the aqueous layer a chloroform/methanol/water-soluble glycophosphosphingolipid fraction. Alkaline degradation and purification by gel filtration chromatography resulted in a tetrasaccharide (phosphatidylinositol (PI)-oligosaccharide A), and a pentasaccharide (PI-oligosaccharide B), each containing 2 mol of 2-aminoethylphosphonate and 1 mol of phosphate. Nuclear magnetic resonance spectroscopy and fast atom bombardment-mass spectrometry suggested that the structure of PI-oligosaccharide A is [formula: see text] and that of PI-oligosaccharide B is as shown. [formula: see text] Both compounds contain an inositol unit linked to ceramide via a phosphodiester bridge. The major aliphatic components of the ceramide portion are stearic acid, lignoceric acid, and C20-phytosphingosine. These novel glycolipids fall within the glycosylated phosphatidylinositol (GPI) family, since they contain the core structure Man alpha (1-->4)GlcNH2 alpha (1-->6)myo-inositol-1-PO4, which is also found in the glycoinositolphospholipids and lipophosphoglycan of Leishmania spp., the L. major promastigote surface protease, the glycosylphosphatidylinositol anchor of Trypanosoma brucei variant surface glycoprotein, and the lipopeptidophosphoglycan of Trypanosoma cruzi. The glycophosphosphingolipids of Leptomonas have features in common with the glycolipids of both Leishmania and T. cruzi, resembling the former by the alpha (1-->3) linkage of mannose to the GPI core, while the 2-aminoethylphosphonate substituent on O-6 of glucosamine and the presence of ceramide in place of glycerol lipids is more reminiscent of T. cruzi. Thus these data lend some support to the hypothesis that both T. cruzi and Leishmania evolved from a Leptomonas-like ancestor.     

Trafficking of Leishmania donovani promastigotes in non-lytic compartments in neutrophils enables the subsequent transfer of parasites to macrophages

Inoculation of Leishmania ( L.) spp. promastigotes in the dermis of mammals by blood-feeding sand flies can be accompanied by the rapid recruitment of neutrophils, inflammatory monocytes and dendritic cells. Despite the presence of these lytic leucocytes, parasitism is efficiently established. We show here that Leishmania donovani promastigotes are targeted to two different compartments in neutrophils. The compartments harbouring either damaged or non-damaged parasites were characterized at the electron microscopy (EM) level using the glucose 6-phosphatase cytochemistry and endosome–phagosome fusion assays. One involves the contribution of lysosomes leading to the formation of highly lytic compartments where parasites are rapidly degraded. The other is lysosome-independent and involves the contribution of a compartment displaying some features of the endoplasmic reticulum (ER) where parasites are protected from degradation. Using genetically modified parasites, we show that the promastigote surface lipophosphoglycan (LPG) is required to inhibit lysosome fusion and maintain parasites in neutrophil compartments displaying ER features. L. donovani -harbouring neutrophils that eventually enter apoptosis can be phagocytosed by macrophages enabling the stealth entry of parasites into their final replicative host cells. Thus, the ability of L. donovani to avoid trafficking into lysosomes-derived compartments in short-lived neutrophils constitutes a key process for the subsequent establishment of long-term parasitism.     

Lysophosphatidylglycerol stimulates chemotactic migration in human natural killer cells

We observed that lysophosphatidylglycerol (LPG) stimulates chemotactic migration in human natural killer (NK) cells. The LPG-induced chemotactic migration of NK cells was completely inhibited by pertussis toxin (PTX). LPG also stimulated the extracellular signal-regulated kinase (ERK) and Akt activities in NK cells. LPG-stimulated ERK activity was inhibited by PTX, indicating the involvement of PTX-sensitive G-proteins. The preincubation of NK cells with an ERK inhibitor (PD98059) or phosphoinositide-3-kinase (PI3K) inhibitors (wortmannin and LY294002) completely inhibited LPG-induced chemotactic migration, suggesting the essential role of ERK and PI3K in the process. Moreover, LPG-induced chemotactic migration in NK cell was inhibited by Ki16425, an LPA_1/3 receptor-selective antagonist, suggesting the involvement of the Ki16425-sensitive G-protein coupled receptor (GPCR) in the process. Taken together, the results indicate that LPG stimulates chemotactic migration in NK cells through GPCR, suggesting a new function of LPG as a modulator of NK cell functioning.     

Intracellular glycosylphosphatidylinositols accumulate on endosomes: toxicity of alpha-toxin to Leishmania major

Glycosylphosphatidylinositols (GPIs) are ubiquitous glycolipids in eukaryotes. In the protozoan Leishmania major, GPIs occur "free" or covalently linked to proteins (e.g., gp63) and polysaccharides. While some free GPIs are detected on the plasma membrane, specific sites where GPIs accumulate intracellularly are unknown in most cells, although the glycolipids are synthesized within the secretory system. Herein, we describe a protocol for identifying intracellular sites of GPI accumulation by using alpha-toxin (from Clostridium septicum). Alpha-toxin bound to gp63 and GPIs from L. major. Intracellular binding sites for alpha-toxin were determined in immunofluorescence assays after removal of GPI-anchored macromolecules (e.g., gp63) from the plasma membrane of fixed cells by using detergent. Endosomes were a major site for GPI accretion in L. major. GPI-less gp63 was detected at the endoplasmic reticulum. In studies with live parasites, alpha-toxin killed L. major with a 50% lethal concentration of 0.77 nM.     

Developmental modification of lipophosphoglycan during the differentiation of Leishmania major promastigotes to an infectious stage.

Protozoan parasites of the genus Leishmania produce the novel surface glycoconjugate, lipophosphoglycan (LPG), which is required for parasite infectivity. In this study we show that LPG structure is modified during the differentiation of L. major promastigotes from a less infectious form in logarithmic growth phase to a highly infectious 'metacyclic' form during stationary growth phase. In both stages, the LPGs comprise linear chains of phosphorylated oligosaccharide repeat units which are anchored to the membrane via a glycosyl-phosphatidylinositol glycolipid anchor. During metacyclogenesis there is (i) an approximate doubling in the average number of repeat units per molecule from 14 to 30, (ii) a pronounced decrease in the relative abundance of repeat units with side chains of beta Gal or Gal beta 1-3Gal beta 1-, and a corresponding increase in repeat units with either no side chains or with side chains of Arap alpha 1-2 Gal beta 1- and (iii) a decrease in the frequency with which the glycolipid anchor is substituted with a single glucose alpha 1-phosphate residue. While the majority of the LPG phosphoglycan chains are capped with the neutral disaccharide, Man alpha 1-2Man, a significant minority of the chains appeared to terminate in non-phosphorylated repeat units and may represent incompletely capped species. We suggest that the developmental modification of LPG may be important in modulating the binding of promastigotes to receptors in the sandfly midgut and on human macrophages and in increasing the resistance of metacyclic promastigotes to complement-mediated lysis.     

Role of exogenous inositol and phosphatidylinositol in glycosylphosphatidylinositol anchor synthesis of GP49 by Giardia lamblia

Although Giardia lamblia trophozoites are unable to carry out de novo phospholipid synthesis, they can assemble complex glycophospholipids from simple lipids and fatty acids acquired from the host. Previously, we have reported that G. lamblia synthesizes GP49, an invariant surface antigen with a glycosylphosphatidylinositol (GPI) anchor. It is therefore possible that myo-inositol (Ins), phosphatidylinositol (PI) and other GPI precursors are obtained from the dietary products of the human small intestine, where the trophozoites colonize. In this report, we have investigated the role of exogenous Ins and PI on GPI anchor synthesis by G. lamblia. The results demonstrate that [(3)H]Ins and PI internalized by trophozoites, metabolically transformed into GlcN(acyl)-PI and downstream GPI molecules. Further investigations suggest that G. lamblia expresses cytidine monophosphate (CMP)-dependent (Mg(2+)-stimulated) and independent (Mn(2+)-stimulated) inositol headgroup exchange enzymes, which are responsible for exchanging free Ins with cellular PI. We observed that 3-deoxy-3-fluoro-D-myo-inositol (3-F-Ins) and 1-deoxy-1-F-scyllo-Ins (1-F-scyllo-Ins), which are considered potent inhibitors of Mn(2+)-stimulated headgroup exchange enzyme, inhibited the incorporation of [(3)H]Ins into PI and GPI molecules significantly, suggesting that CMP-independent (Mn(2+)-stimulated) exchange enzyme may be important for these reactions. However, 3-F-Ins and 1-F-scyllo-Ins were not effective in blocking the incorporation of exogenously supplied [(3)H]PI into GPI glycolipids. Thus, it can be concluded that G. lamblia can use exogenously supplied [(3)H]PI and [(3)H]Ins to synthesize GPI glycolipids of GP49; while PI is directly incorporated into GPI molecules, free Ins is first converted into PI by headgroup exchange enzymes, and this newly formed PI participates in GPI anchor synthesis.     

Biosynthesis of glycolipid precursors for glycosylphosphatidylinositol membrane anchors in a Toxoplasma gondii cell-free system.

Toxoplasmosis, a disease that affects humans and a wide variety of mammals is caused by Toxoplasma gondii, the obligate intracellular coccidian protozoan parasite. Most T. gondii research has focused on the rapidly growing invasive form, the tachyzoite, which expresses five major surface proteins attached to the parasite membrane by glycosylphosphatidylinositol (GPI) anchors. We have recently reported the purification and partial characterization of candidate precursor glycolipids (GPIs) from metabolically labeled parasites and have presented evidence that these GPIs have a linear glycan backbone sequence indistinguishable from the GPI core glycan of the major tachyzoite surface protein, P30. In this report, we describe a cell-free system derived from tachyzoite membranes which is capable of catalyzing GPI biosynthesis. Incubation of the membrane preparations with radioactive sugar nucleotides (GDP-[3H]mannose or UDP-[3H]GlcNAc) resulted in incorporation of radiolabeled into numerous glycolipids. By using a combination of chemical/enzymatic tests and chromatographic analysis, a series of incompletely glycosylated lipid species and mature GPIs have been identified. We have also established the involvement of Dol-P-mannose in the synthesis of T. gondii GPIs by demonstrating that the incorporation of [3H]mannose into the mannosylated GPIs is stimulated by dolichylphosphate and inhibited by amphomycin. In addition, increasing the concentration of nonradioactive GDP mannose resulted in a loss of radiolabel from the first easily detectable GPI precursor, GlcN-PI, and a concomittant appearance of the radio-activity into mannosylated glycolipids. Altogether, our data suggest that the GPI core glycan in T. gondii is assembled via sequential glycosylation of phosphatidylinositol, as proposed for the biosynthesis of GPIs in Trypanosoma brucei. In contrast to T. brucei, preliminary experiments indicate that the core glycan of some GPIs synthesized by the T. gondii cell-free system is modified by N-acetylgalactosamine similar to the situation for mammalian Thy-1.     

Structural analysis of a glycosylphosphatidylinositol glycolipid ofLeishmania donovani

A glycosylphosphatidylinositol (GPI) glycolipid antigen recognized by sera from patients with visceral leishmaniasis was isolated from Leishmania donovani promastigotes. The carbohydrate moiety was cleaved from the lipid part by digestion with specific phosphatidylinositol phospholipase C. After separation, structural analysis was carried out on the phosphorylated inositol oligosaccharide and the alkylacyl glycerol. The following major structures were found: [formula: see text] The presence of the conserved sequence Man alpha 1-2Man alpha 1-6Man alpha 1-4GlcN-PI of glycosyl phosphatidylinositol protein anchors in this antigen may be consistent with a precursor role of Leishmania glycosyl phosphatidylinositol anchored proteins for this glycolipid.     

Leishmania mexicana: participation of NF-kappaB in the differential production of IL-12 in dendritic cells and monocytes induced by lipophosphoglycan (LPG)

Dendritic cells (DC) and macrophages (Mφ) are well known as important effectors of the innate immune system and their ability to produce IL-12 indicates that they possess the potential of directing acquired immunity toward a Th1-biased response. Interestingly, the intracellular parasite Leishmania has been shown to selectively suppress Mφ IL-12 production and are DC the principal source of this cytokine. The molecular details of this phenomenon remain enigmatic. In the present study we examined the effect of Leishmania mexicana lipophosphoglycan (LPG) on the production of IL-12, TNF-α, and IL-10 and nuclear translocation of NF-κB. The results show that LPG induced more IL-12 in human DC than in monocytes. This difference was due in part to nuclear translocation of NF-κB, since LPG induced more translocation in DC than in monocytes. These results suggest that Leishmania LPG impairs nuclear translocation of NF-κB in monocytes with the subsequent decrease in IL-12 production.     

Inhibition of phosphatidylinositol 4-kinase results in a significant reduced respiratory burst in formyl-methionyl-leucyl-phenylalanine-stimulated human neutrophils

The effects of phenylarsine oxide and a monoclonal antibody directed against type II phosphatidylinositol 4-kinase (PI4K) on the N-formyl-methionyl-leucyl-phenylalanine (fMLP)-stimulated respiratory burst and the PI4K activity in neutrophils were investigated. Fluorescence microscopic imaging showed that the antibody labeled with IANBD amide (N,N'-dimethyl-N-(iodoacetyl)-N'-(7-nitrobenz-2-oxa-1,3-diazol-4-yl)ethylenediamine) could enter into the cytosol possibly by endocytosis. It was found that the antibody inhibited the fMLP-stimulated respiratory burst but had little effect on the phorbol myristate acetate-activated respiratory burst in neutrophils, whereas phenylarsine oxide inhibited both. It was found that even at higher concentration, the antibody could not completely inhibit the cell response. Using cells preincubated with human immunoglobulin G of the same concentration as the control, the maximal inhibition of the fMLP-stimulated respiratory burst by the antibody against type II PI4K was found to be about 70%, whereas the PI4K activity was inhibited by only about 40%. The discrepancy in depressing the cell response and the enzyme activity may be the result of depletion of the phosphatidylinositol 4,5-bisphosphate or phosphatidylinositol 3,4,5-trisphosphate pools during the incubation of cells with the antibody. Both the 40% inhibition of PI4K activity and 70% depression of the respiratory burst by the type II PI4K antibody may imply that at least 40% of the phosphatidylinositol 4,5-biphosphate was synthesized promptly by all forms of PI4K and phosphatidylinositol-4-phosphate 5-kinase in the fMLP-activated cells. The results suggest that PI4K plays a central role in either phospholipase C or PI3K signaling and that PI3K, PI4K, and phosphatidylinositol 4-phosphate 5-kinase must be considered as an integrated family for the phosphatidylinositol 3,4,5-trisphosphate initiated signaling.