Superoxide anion participation in human monocyte-mediated oxidation of low-density lipoprotein and conversion of low-density lipoprotein to a cytotoxin

Human monocytes, upon activation with opsonized zymosan, altered low-density lipoprotein (LDL) during a 24-h co-incubation, resulting in its oxidation and acquisition of cytotoxic activity against target fibroblast cell lines. Both the oxidation of LDL and its conversion to a cytotoxin were enhanced with time of incubation, with the most substantial changes occurring after 6 h of culture of LDL with activated monocytes. Unactivated monocytes did not mediate either alteration. Superoxide anion (O2-) participated in both the oxidation of LDL and its conversion to a cytotoxin since addition of superoxide dismutase (SOD) at the beginning of the co-incubation inhibited, in a concentration dependent fashion, both the monocyte-mediated oxidation and the monocyte-mediated conversion of LDL to a cytotoxin. As expected, the rate of superoxide anion release was greatest during the respiratory burst, very early in the 24-h incubation (0 to 2 h); however, exposure of LDL to monocytes during the respiratory burst was not required for LDL oxidation. The lower levels of O2- released by the cells hours after the respiratory burst had subsided were sufficient to lead to the initiation of LDL oxidation. Three results indicated that the oxidative modification of LDL into a cytotoxin required O2(-)-independent free radical propagation after O2(-)-dependent initiation. First, oxidation of LDL exposed to the activated, superoxide anion-releasing monocytes for 6 h could be almost completely blocked by the addition at 6 h of the general free radical scavenger butylated hydroxytoluene, but not by SOD. Second, LDL oxidation proceeded even after removal of LDL from the superoxide anion-producing, activated cells after various durations of exposure. Third, the development of substantial levels of lipid peroxidation products and the development of greater cytotoxicity occurred after 6 h of exposure of LDL to activated cells, long after peak O2- release had subsided. These results lead us to conclude that monocyte-mediated oxidation of LDL, leading to its transformation into a cytotoxin, requires release of O2- occurring as a result of activation but not necessarily during the respiratory burst, and also requires O2(-)-independent free radical propagation. The modification of LDL into a potent toxin by activated monocytes may explain the tissue damage in atherosclerotic lesions and other pathologic sites in which inflammatory cells congregate.     

Human apo-lipoprotein B from normal plasma contains oxidised peptides

Oxidation of low density lipoprotein (LDL) may be atherogenic, but radical-initiated oxidation of its apoprotein B-100 (apoB) has been little studied. Transition metal ions iron and copper are candidates for mediating radical oxidation of LDL in vivo. Therefore, we studied the copper-ion-induced oxidation of apoB in human LDL. Using HPLC methods developed in our recent work, we studied the destruction of native and the generation of six oxidised amino acids; we also assessed the release of peptides from the LDL particle by FPLC. We observed time-dependent losses of apoB histidine, lysine and glycine. Long-lived reactive species, the reductant DOPA, and the oxidant hydroperoxides of valine and leucine (measured as hydroxides after reduction), were generated. Their relative abundance (mol/mol of parent amino acid) was DOPA>o- and m-tyrosine>dityrosine, valine-hydroxides, leucine hydroxides. Low molecular weight fragments were also released from the LDL in a time-dependent manner, contained hydroperoxides sensitive to GSH peroxidase, and generated radicals on reaction with iron–EDTA. The fragments contained peptides active in the quinone redox cycling procedure, comprising 0.25% of the supplied LDL amino acids. Characteristic peptides were present in each FPLC fraction containing the fragments, as judged by further HPLC fractionation. Some fragments were present in the unoxidised LDL preparations, and when these were largely removed by FPLC, copper oxidation could still generate fragments, suggesting that those present in the starting material might indicate prior oxidation. Concordantly, we found that fresh plasma LDL apoB contained 3% of total plasma protein-bound oxidised amino acids, and with the same relative abundance. We conclude that plasma proteins including apoB are subject to physiological oxidation, similar to that inflicted by copper ions; the latter may contribute to intimal LDL oxidation, which could be the source of oxidised plasma apoB.     

Ceruloplasmin and cardiovascular disease

Transition metal ion–mediated oxidation is a commonly used model system for studies of the chemical, structural, and functional modifications of low-density lipoprotein (LDL). The physiological relevance of studies using free metal ions is unclear and has led to an exploration of free metal ion-independent mechanisms of oxidation. We and others have investigated the role of human ceruloplasmin (Cp) in oxidative processes because it the principal copper-containing protein in serum. There is an abundance of epidemiological data that suggests that serum Cp may be an important risk factor predicting myocardial infarction and cardiovascular disease. Biochemical studies have shown that Cp is a potent catalyst of LDL oxidation in vitro. The pro-oxidant activity of Cp requires an intact structure, and a single copper atom at the surface of the protein, near His⁴²⁶, is required for LDL oxidation. Under conditions where inhibitory protein (such as albumin) is present, LDL oxidation by Cp is optimal in the presence of superoxide, which reduces the surface copper atom of Cp. Cultured vascular endothelial and smooth muscle cells also oxidize LDL in the presence of Cp. Superoxide release by these cells is a critical factor regulating the rate of oxidation. Cultured monocytic cells, when activated by zymosan, can oxidize LDL, but these cells are unique in their secretion of Cp. Inhibitor studies using Cp-specific antibodies and antisense oligonucleotides show that Cp is a major contributor to LDL oxidation by these cells. The role of Cp in lipoprotein oxidation and atherosclerotic lesion progression in vivo has not been directly assessed and is an important area for future studies.     

Oxidized low density lipoprotein inhibits the hemolytic activity of Asp-hemolysin from Aspergillus fumigatus

We have examined the effect of chemically modified human low density lipoproteins (LDLs) , acetylated LDL and oxidized LDL, on the hemolytic activity of Asp-hemolysin. Oxidized LDL, but not acetylated LDL, inhibited the hemolytic activity of this toxin. The inhibitory effects of oxidized LDL increased with the time of Cu²⁺-induced LDL oxidation. Similar inhibition was observed in the filtrate which was separated from the incubation mixture of Asp-hemolysin with oxidized LDL (for 2 h of oxidation) following ultrafiltration through a membrane with a molecular mass cutoff of 100 000. However, at longer LDL oxidation times, the inhibition by the filtrates was less than the control mixture without ultrafiltration. We suggest that the inhibition by oxidized LDL was due to the binding of oxidized LDL to Asp-hemolysin at shorter LDL oxidation times .     

Inhibition of luminol-dependent luminescence and simultaneous generation of native luminescence of activated human polymorphonuclear leukocytes by addition of albumin

Luminol-dependent luminescence (LDL) and luminol-independent, native luminescence (NL) of polymorphonuclear leukocytes were investigated with respect to the effects generated by the addition of albumin to the reaction medium. The cells were activated: (1) by simple surface attachment to a hydrophilic plastic, (2) by opsonized zymosan, (3) by phorbol myristate acetate, (4) by formylmethionyl-leucyl-phenylalaline. Both kinds of emissions were recorded simultaneously using a method of spectral discrimination. The addition of albumin resulted in an inhibition of LDL, which coincided with a generation of NL. The extent of the inhibition of LDL depended on the type of stimulus used. Maximum inhibition occurred with cells activated by attachment to plastic surfaces and minimum inhibition was observed with cells stimulated by opsonized zymosan. Different contributions of extracellularly released reactive oxygen-species may be responsible for this. It appears possible to discriminate between intra- and extracellular sites of oxygen-metabolites production using albumin simultaneously as extracellular quencher of LDL and as luminescent probe for NL.     

Cell-mediated LDL oxidation: the impact of transition metals and transferrin

Activated monocytes release oxygen radicals by respiratory burst and oxidative damage can be accelerated by transition metals. We investigated the cell-mediated and metal-catalysed in vitro oxidation of low-density lipoproteins (LDL), as well as the impact of the metal-binding protein transferrin (Tf). LDL oxidation was measured by monitoring the increase in fluorescence (350/440 nm excitation/emission). Maximal respiratory burst by U937 cells was achieved after 96 h differentiation with retinoic acid and dihydroxyvitamin D3 followed by stimulation with opsonised zymosan. Addition of activated cells resulted in the LDL oxidation, even in the absence of transition metals. Moreover, activated cells greatly enhanced metal-catalysed oxidative modifications, especially in the presence of copper. By binding metals, Tf was able to strongly impair this process. In conclusion, by generating oxygen radicals, activated U937 cells were able to oxidise LDL. The oxidising process was most pronounced in the presence of copper and could be blocked by Tf.     

Minimally oxidized LDL inhibits macrophage selective cholesteryl ester uptake and native LDL-induced foam cell formation

Scavenger receptor-mediated uptake of oxidized LDL (oxLDL) is thought to be the major mechanism of foam cell generation in atherosclerotic lesions. Recent data has indicated that native LDL is also capable of contributing to foam cell formation via low-affinity receptor-independent LDL particle pinocytosis and selective cholesteryl ester (CE) uptake. In the current investigation, Cu²⁺-induced LDL oxidation was found to inhibit macrophage selective CE uptake. Impairment of selective CE uptake was significant with LDL oxidized for as little as 30 min and correlated with oxidative fragmentation of apoB. In contrast, LDL aggregation, LDL CE oxidation, and the enhancement of scavenger receptor-mediated LDL particle uptake required at least 3 h of oxidation. Selective CE uptake did not require expression of the LDL receptor (LDL-R) and was inhibited similarly by LDL oxidation in LDL-R^−/− versus WT macrophages. Inhibition of selective uptake was also observed when cells were pretreated or cotreated with minimally oxidized LDL, indicating a direct inhibitory effect of this oxLDL on macrophages. Consistent with the effect on LDL CE uptake, minimal LDL oxidation almost completely prevented LDL-induced foam cell formation. These data demonstrate a novel inhibitory effect of mildly oxidized LDL that may reduce foam cell formation in atherosclerosis.     

Scavenging of Lipid Peroxidation Products from Oxidizing LDL by Albumin Alters the Plasma Half-Life of a Fraction of Oxidized LDL Particles

We analyse LDL oxidation in vitro in the presence of copper (II) ions and differentiate a lag phase and a rapid peroxidation phase. We demonstrate that a physiological concentration of albumin does not alter the kinetics of the dienes in the oxidizing LDL but reduces the fluorescence of the oxidizing LDL and alters the biological properties of oxidized LDL. We find in rats after intravenous administration of oxidized LDL, that it is rapidly cleared from the circulating blood. The presence of albumin during the peroxidation phase, however, reduces the fraction of oxidized LDL with rapid blood clearance. We propose that some lipid peroxidation products formed in oxidizing LDL are hydrophilic enough to diffuse into the aqueous buffer from where they react either with the s-amino-groups of apolipoprotein B or albumin. Effective scavengers for these hydrophilic endproducts of the LDL oxidation pathways such as albumin might reduce modification of the LDL and might be useful to reduce its atherogenicity.     

Scavenging of Lipid Peroxidation Products from Oxidizing LDL by Albumin Alters the Plasma Half-Life of a Fraction of Oxidized LDL Particles

We analyse LDL oxidation in vitro in the presence of copper (II) ions and differentiate a lag phase and a rapid peroxidation phase. We demonstrate that a physiological concentration of albumin does not alter the kinetics of the dienes in the oxidizing LDL but reduces the fluorescence of the oxidizing LDL and alters the biological properties of oxidized LDL. We find in rats after intravenous administration of oxidized LDL, that it is rapidly cleared from the circulating blood. The presence of albumin during the peroxidation phase, however, reduces the fraction of oxidized LDL with rapid blood clearance. We propose that some lipid peroxidation products formed in oxidizing LDL are hydrophilic enough to diffuse into the aqueous buffer from where they react either with the s-amino-groups of apolipoprotein B or albumin. Effective scavengers for these hydrophilic endproducts of the LDL oxidation pathways such as albumin might reduce modification of the LDL and might be useful to reduce its atherogenicity.     

Requirement of lipophosphoglycan for intracellular survival of Leishmania donovani within human monocytes

The function of the lipophosphoglycan of Leishmania donovani parasites was investigated in human peripheral monocytes. In contrast to wild-type L. donovani which grow in monocytes, incubation of monocytes with two variant lines of L. donovani, defective in lipophosphoglycan expression, resulted in the entry of the variant cells into the monocytes and their subsequent destruction. Passive transfer of lipophosphoglycan to the variant cells led to prolonged survival in monocytes. These results indicate that lipophosphoglycan is required by the parasite for intracellular survival. To investigate one possible protective role of the glycoconjugate, preincubation of monocytes with a suspension of lipophosphoglycan and subsequent treatment of the cells with PMA or opsonized zymosan resulted in an attenuation of the oxidative burst; the attenuation effect was concentration dependent on the glycoconjugate and independent of preincubation time. Moreover, hydrophobic beads, coated with lipophosphoglycan, were phagocytized by monocytes and found to inhibit oxygen consumption in monocytes activated with PMA. These results suggest a possible relationship between the absence of lipophosphoglycan in the variant parasites and their inability to survive within monocytes. Although the precise molecular basis remains to be elucidated, the ability of lipophosphoglycan to impair the microbial oxidative response may be a contributing factor in its requirement for intracellular survival.     

Thrombin enhances opsonized zymosan-induced chemiluminescence of neutrophils

We examined the effects of alpha-thrombin (the native enzyme) on neutrophil activation as assessed by the measurement of chemiluminescence. alpha-Thrombin in physiological concentrations (10(-9)-10(-8)M) did not induce neutrophil chemiluminescence. However, when neutrophils were coincubated with opsonized zymosan and alpha-thrombin, the chemiluminescence response to opsonized zymosan was enhanced in a concentration-dependent manner. The neutrophil chemiluminescence responses to opsonized zymosan and to opsonized zymosan plus alpha-thrombin were dependent on the generation of oxygen-derived free radicals since the chemiluminescence was inhibited by superoxide dismutase. The results indicate that thrombin per se does not induce neutrophil chemiluminescence. However, thrombin enhances the chemiluminescence response to opsonized zymosan suggesting an interaction between thrombin and complement receptors in inducing neutrophil activation. The chemiluminescence response to thrombin and opsonized zymosan is the result of oxygen-derived free radicals.     

Glucose-Modified Low Density Lipoprotein Enhances Human Monocyte Chemotaxis

In diabetes mellitus the progression of atherosclerosis is accelerated. The interaction of glucose with athero-genic lipoproteins may be relevant to the mechanisms responsible for this vascular damage. The aim of this study was to examine the effect of glucose-modified low density lipoprotein (LDL) on human monocyte chemotaxis and to investigate the roles of oxidation and glycation in the generation of chemotactic LDL. Cu(II)-mediated LDL oxidation was potentiated by glucose in a dose-dependent manner and increased its chemotactic activity. Incubation with glucose alone, under conditions where very little oxidation was observed, also increased the chemotactic property of LDL. Neither diethylenetriamine pentaacetic acid (DETAPAC) nor aminoguanidine, which both inhibited LDL oxidation, completely inhibited the chemotactic activity of glycated oxidised LDL. The results suggest that both oxidation and glycation contribute to increased chemotactic activity.     

Pentoxifylline Pretreatment Enhances the Oxidative Burst Activity of Human Peripheral Blood Mononuclear Cells

The effect of pentoxifylline pretreatment on the lucigenin-augmented chemiluminescence and dismutase-inhibitable superoxide production of human neutrophils and mononuclear cells (MNCs) was studied. Pentoxifylline at 20–2,000 μg/ml enhanced the lucigenin-augmented chemiluminescence (118–165% of the control, P < 0.01) of phorbol myristate acetate (PMA)-stimulated MNC. Pentoxifylline at 20–2,000 μg/ml increased the MNC superoxide production, i.e., 142–171% of the control (P < 0.05) using PMA stimulation and 145–159% of the control (P < 0.01) using opsonized zymosan stimulation. In contrast, pentoxifylline (up to 2,000 μg/ml) did not influence the lucigenin-augmented chemiluminescence and superoxide production of human neutrophils, stimulated by either PMA or opsonized zymosan. These results suggest that pentoxifylline is an immunomodulator and may have potential usefulness in the enhancement of immune defenses in compromised hosts.     

Cellular Thiol Production and Oxidation of Low-Density Lipoprotein

Compelling evidence suggests that low-density lipoprotein (LDL) is oxidized by cells within the arterial intima and that, once oxidized, it is profoundly atherogenic. The precise mechanism(s) by which cells promote the oxidation of LDL in vivo are not known; in vitro, however, oxidation of LDL can be enhanced by a number of differing mechanisms, including reaction with free and protein-bound metal ions, thiols, reactive oxygen species, lipoxygenase, myeloperoxidase and peroxynitrite. This review is concerned with the mechanisms by which cells enhance the oxidation of LDL in the presence of transition metals; in particular, the regulation, pro- and anti-oxidant consequences, and mechanism of action of cellular thiol production are examined, and contrasted with thiol-independent oxidation of LDL in the presence of transition metals.     

Protein oxidation by the cytochrome P450 mixed-function oxidation system

This mini-review summarizes results of studies on the oxidation of proteins and low-density lipoprotein (LDL) by various mixed-function oxidation (MFO) systems. Oxidation of LDL by the O₂/FeCl₃/H₂O₂/ascorbate MFO system is dependent on all four components and is much greater when reactions are carried out in the presence of a physiological bicarbonate/CO₂ buffer system as compared to phosphate buffer. However, FeCl₃ in this system could be replaced by hemin or the heme-containing protein, hemoglobin, or cytochrome c. Oxidation of LDL by the O₂/cytochrome P450 cytochrome c reductase/NADPH/FeCl₃ MFO system is only slightly higher (25%) in the bicarbonate/CO₂ buffer as compared to phosphate buffer, but is dependent on all components except FeCl₃. Omission of FeCl₃ led to a 60% loss of activity. These results suggest that peroxymonobicarbonate and/or free radical derivatives of bicarbonate ion and/or CO₂ might contribute to LDL oxidation by these MFO systems.     

Lysosomal enzyme release from human monocytes in response to particulate stimuli

Lysosomal enzyme release from human monocytes was evaluated in response to opsonized zymosan, opsonized sheep erythrocytes, and latex beads. Monocytes were found to release lysosomal enzymes immediately upon challenge with all three phagocytosable particles. Cytochalasin B enhanced beta-glucosaminidase release from mononuclear cells challenged with opsonized zymosan or opsonized red blood cells, but inhibited the response to latex particles. Lysosomal enzyme release was found to be independent of protein synthesis, and in the absence of cytochalasin B required the stimulus to be presented either as a phagocytosable particle or immobilized on a surface. The kinetics of enzyme release and phagocytosis were also examined and found to be different, lending support to the hypothesis that lysosomal enzyme release may be a physiologic response to a biologic stimulus in vivo and not simply an "accidental" consequence of an ongoing phagocytic event.     

Phospholipid and arachidonic acid metabolism in zymosan-stimulated human monocytes: modulation by cAMP

Receptor-ligand interaction in mononuclear phagocytes is intimately linked to alterations in membrane phospholipids and release of arachidonic acid (AA). In addition, synthesis of bioactive lipids from released AA can result in further modification of cell responses. Upon challenge with opsonized zymosan, [3H]-arachidonic acid ([3H]-AA)-labeled human monocytes released 25 +/- 2% of their incorporated radiolabel within 30 min. Pretreatment of the monocytes with 5 X 10(-4) M isobutylmethylxanthine (IBMX) or 1 X 10(-3) M dibutyryl cyclic AMP (d-cAMP) inhibited total [3H]-AA release in the presence of zymosan by 47% and 42%, respectively. Analysis of incorporated [3H]-AA in cellular phospholipid pools indicated that significant amounts of label were lost from both phosphatidylcholine (PC) and phosphatidylinositol (PI) during zymosan stimulation. Treatment with d-cAMP substantially inhibited the loss of label from PC, but had no affect on PI. HPLC analysis of cell supernatants from zymosan-treated cells indicated that 5-HETE was the predominant metabolite generated from [3H]-AA, and its production was depressed during treatment with d-cAMP. Phospholipase activity in human monocyte homogenates was not effected by d-cAMP or IBMX at the highest concentrations used, whether these were added directly to the homogenate or by pretreatment of whole cells, demonstrating that inhibition required an intact cell. These results suggest that human monocytes exposed to opsonized zymosan release AA via two mechanisms and that modulation by cAMP is indirectly effecting a phospholipase directed towards PC.     

Edta Differentially and Incompletely Inhibits Components of Prolonged Cell-Mediated Oxidation of Low-Density Lipoprotein

The extent to which cells can oxidize LDL may be underestimated because of the use of standard and arbitrary 24 hour in vitro incubations of cells with LDL. Such incubations have resulted in inconsistent results regarding the ability of cell-mediated LDL oxidation to generate relatively advanced oxidation products such as 7-ketocholesterol (7-KC). We studied prolonged oxidation of low density lipoprotein (LDL) by mouse peritoneal macrophages using HPLC measurement of cholesterol, cholesteryl esters and their oxidation products 7-KC and cholesteryl linoleate hydroperoxide (CL-OOH). Cell-mediated oxidation in Ham's F10 consistently followed the successive stages previously described during 24 hour-10 μM copper-mediated LDL oxidation, always generating 7-KC if allowed to proceed for sufficient time. The degree of inhibition of LDL oxidation achieved by metal chelators EDTA and DTPA at more advanced stages of cell-mediated LDL oxidation was not predictable from the published effects of such chelators upon early stages of metal-mediated and cell-mediated LDL oxidation. EDTA and DTPA only incompletely prevented the consumption of cholesteryl esters and the loss of preformed CL-OOH when added after cell-mediated LDL oxidation was established, while effectively concurrently inhibiting the generation of 7-KC. These data indicate that progressive cell-mediated peroxidation of LDL cholesteryl esters and decomposition of CL-OOH may be less dependent upon a continuing supply of redox active metals than is the generation of 7-KC. In addition, they confirm the plausibility of prolonged cell-mediated oxidation of LDL as a source of oxysterols found in human atherosclerotic plaque, and imply that active redox cycling of metals is particularly important for their generation in vivo.     

High‐density lipoprotein acts as an opsonin to enhance phagocytosis of group A streptococcus by U937 cells

We have previously demonstrated that high‐density lipoprotein (HDL) can specifically bind to streptococcal collagen‐like protein 1 (Scl1) of M41‐type group A Streptococcus (GAS). However, the pathological or physiological significance of Scl1?HDL interaction is unknown. Here, the hypothesis that HDL acts as an opsonin to enhance phagocytosis of HDL‐bound GAS by monocytes given that some scavenger receptors can mediate the endocytosis of HDL was tested by using FITC‐labeled bacteria, human U937 monocytes and HDL for phagocytic assays. HDL (10 µg/mL) was found to significantly enhance internalization of M41‐type (ATCC 12373) GAS by U937 cells after 60 min incubation, compared with an HDL‐free group. The internalized GAS were dead after 60 min incubation with U937 cells regardless of presence and absence of HDL. Although very‐low‐density lipoprotein (VLDL) could specifically bind to ATCC 12373 strain, it did not promote phagocytosis of GAS. Additionally, LDL, HDL and VLDL did not enhance phagocytosis of CMCC 32198 strain because this strain did not bind to these lipoproteins. A physiological concentration of HDL (1000 µg/mL) had a similar effect. Anti‐CD36 antibody completely abolished opsonic phagocytosis whereas anti‐CD4 antibody did not, indicating that CD36 is the major scavenger receptor mediating the uptake of HDL‐opsonized GAS by U937 cells. Furthermore, because rScl1 competitively blocked the interaction of ATCC 12373 strain with HDL recombinant Scl1 (rScl1) derived from M41‐type GAS, it significantly decreased opsonophagocytosis of ATCC 12373 strain but not of CMCC 32198 strain. Therefore, our findings suggest that HDL may be an opsonin that enhances CD36‐dependent opsonophagocytosis of GAS by U937 cells.

     

Effect of botulinum D toxin on neutrophils

Activated botulinum D toxin ADP-ribosylates a 22 kDa molecular weight protein in homogenates obtained by sonication of a suspension of rabbit peritoneal neutrophils. The ADP-ribosylation catalyzed by activated botulinum D toxin is inhibited in homogenates obtained from cells pretreated with the toxin, suggesting that it is able to enter into these cells and be activated by them. The rise in intracellular concentration of free calcium in toxin treated cells stimulated by fMet-Leu-Phe is similar to that found in control cells. The basal concentration of intracellular free calcium is significantly elevated in neutrophils treated with the intact but not with the activated form of the botulinum D toxin. Superoxide generation in control and native toxin treated cells stimulated with fMet-leu-Phe, phorbol 12-myristate 13-acetate or opsonized zymosan is the same. The release of beta-glucosaminidase produced by fMet-Leu-Phe or Concanavalin A in botulinum D toxin treated neutrophils was slightly higher than the corresponding release in control cells. Furthermore, the fMet-Leu-Phe-induced increase in the amount of actin associated with the cytoskeleton is not inhibited by botulinum D toxin. These results suggest that the 22 kDa protein which can be ADP-ribosylated by botulinum D toxin is not involved in these stimulated neutrophil responses.