Congenital neutropenia in a newborn.

Severe congenital neutropenia (SCN) is a genetically heterogeneous, rare disorder defined by a persistent absolute neutrophil count <500k mm(-3) with neutrophil maturation arrest at the promyelocyte stage and an increased risk for infection as well as a propensity towards developing myelodysplastic syndrome and acute myelogenous leukemia. We report a case of incidentally identified SCN in a full-term, otherwise healthy infant girl. Routine complete blood counts obtained for follow up of ABO incompatibility-induced jaundice and anemia identified mild neutropenia at birth followed by severe persistent neutropenia by 1 week of birth. Genetic testing confirmed the clinical suspicion of SCN with the identification of a mutation in the ELANE gene. Prompt identification and treatment of infants with SCN is critical to minimizing morbidity and mortality; as such, a diagnosis of SCN must be considered in all infants with neutropenia even in the absence of infection.

Stem cell transplantation in patients with severe congenital neutropenia with evidence of leukemic transformation.

Severe congenital neutropenia (SCN) is a hematologic condition characterized by arrested maturation of myelopoiesis at the promyelocyte stage of development. With appropriate treatment using recombinant human granulocyte-colony-stimulating factor (r-HuG-CSF), SCN patients are now surviving longer, but are at increased risk of developing myelodysplastic syndrome (MDS)/acute myeloid leukemia (AML). Hematopoietic stem cell transplantation (HSCT) is the only curative option for these patients, but transplantation outcomes after malignant transformation are not well established. We report results for six patients with SCN who underwent HSCT for MDS or AML between 1997 and 2001 at two transplant centers. Two patients transplanted for MDS survived. Both of these patients were transplanted without being given induction chemotherapy. Four patients, who all received induction chemotherapy for AML prior to HSCT, died. Administering induction chemotherapy prior to HSCT resulted in significant morbidity. Rapid transplantation should be the goal for the SCN patient once the diagnosis of MDS/AML is established. SCN patients should be monitored carefully for progression to MDS in order to be treated with HSCT as soon as they have progressed and before developing AML. For SCN patients who progress to AML, HSCT should still be considered, even though the risks appear to be greater.

Characterization of four CD18 mutants in leucocyte adhesion deficient (LAD) patients with differential capacities to support expression and function of the CD11/CD18 integrins LFA-1, Mac-1 and p150,95.

Leucocyte adhesion deficiency (LAD) is a hereditary disorder caused by mutations in the CD18 (beta2 integrin) gene. Four missense mutations have been identified in three patients. CD18(A270V) supports, at a diminished level, CD11b/CD18 (Mac-1, alphaMbeta2 integrin) and CD11c/CD18 (p150,95, alphaXbeta2 integrin) expression and function but not CD11a/CD18 (LFA-1, alphaLbeta2 integrin) expression. Conversely, CD18(A341P) supports a limited level of expression and function of CD11a/CD18, but not of the other two CD11/CD18 antigens. CD18(C590R) and CD18(R593C) show a decreasing capacity to associate with the CD11a, CD11c and CD11b subunits. Transfectants expressing the CD11a/CD18 with the C590R and R593C mutations are more adhesive than transfectants expressing wild-type LFA-1, and express the reporter epitope of the monoclonal antibody 24 constitutively. Thus, the four mutations affect CD18 differently in its capacities to support CD11/CD18 expression and adhesion. These results not only provide a biochemical account for the clinical diversity of patients with leucocyte adhesion deficiency, but also offer novel insights into the structural basis of interaction between the alpha and beta subunits, which is an integral component in our understanding of integrin-mediated adhesion and its regulation.

Mutations in the gene encoding neutrophil elastase in congenital and cyclic neutropenia.

Congenital neutropenia and cyclic neutropenia are disorders of neutrophil production predisposing patients to recurrent bacterial infections. Recently the locus for autosomal dominant cyclic neutropenia was mapped to chromosome 19p13.3, and this disease is now attributable to mutations of the gene encoding neutrophil elastase (the ELA2 gene). The authors hypothesized that congenital neutropenia is also due to mutations of neutrophil elastase. Patients with congenital neutropenia, cyclic neutropenia, or Shwachman-Diamond syndrome were referred to the Severe Chronic Neutropenia International Registry. Referring physicians provided hematologic and clinical data. Mutational analysis was performed by sequencing polymerase chain reaction (PCR)-amplified genomic DNA for each of the 5 exons of the neutrophil ELA2 gene and 20 bases of the flanking regions. RNA from bone marrow mononuclear cells was used to determine if the affected patients expressed both the normal and the abnormal transcript. Twenty-two of 25 patients with congenital neutropenia had 18 different heterozygous mutations. Four of 4 patients with cyclic neutropenia and 0 of 3 patients with Shwachman-Diamond syndrome had mutations. For 5 patients with congenital neutropenia having mutations predicted to alter RNA splicing or transcript structure, reverse transcriptase-PCR showed expression of both normal and abnormal transcripts. In cyclic neutropenia, the mutations appeared to cluster near the active site of the molecule, whereas the opposite face was predominantly affected by the mutations found in congenital neutropenia. This study indicates that mutations of the gene encoding neutrophil elastase are probably the most common cause for severe congenital neutropenia as well as the cause for sporadic and autosomal dominant cyclic neutropenia.

Myelodysplasia syndrome and acute myeloid leukemia in patients with congenital neutropenia receiving G-CSF therapy.

Granulocyte colony-stimulating factor (G-CSF) has had a major impact on management of "severe chronic neutropenia," a collective term referring to congenital, idiopathic, or cyclic neutropenia. Almost all patients respond to G-CSF with increased neutrophils, reduced infections, and improved survival. Some responders with congenital neutropenia have developed myelodysplastic syndrome and acute myeloblastic leukemia (MDS/AML), which raises the question of the role of G-CSF in pathogenesis. The Severe Chronic Neutropenia International Registry (SCNIR), Seattle, WA, has data on 696 neutropenic patients, including 352 patients with congenital neutropenia, treated with G-CSF from 1987 to present. Treatment and patient demographic data were analyzed. The 352 congenital patients were observed for a mean of 6 years (range, 0.1-11 years) while being treated. Of these patients, 31 developed MDS/AML, for a crude rate of malignant transformation of nearly 9%. None of the 344 patients with idiopathic or cyclic neutropenia developed MDS/AML. Transformation was associated with acquired marrow cytogenetic clonal changes: 18 patients developed a partial or complete loss of chromosome 7, and 9 patients manifested abnormalities of chromosome 21 (usually trisomy 21). For each yearly treatment interval, the annual rate of MDS/AML development was less than 2%. No significant relationships between age at onset of MDS/AML and patient gender, G-CSF dose, or treatment duration were found (P >.15). In addition to the 31 patients who developed MDS/AML, the SCNIR also has data on 9 additional neutropenic patients whose bone marrow studies show cytogenetic clonal changes but the patients are without transformation to MDS/AML. Although our data does not support a cause-and-effect relationship between development of MDS/AML and G-CSF therapy or other patient demographics, we cannot exclude a direct contribution of G-CSF in the pathogenesis of MDS/AML. This issue is unclear because MDS/AML was not seen in cyclic or idiopathic neutropenia. Improved survival of congenital neutropenia patients receiving G-CSF therapy may allow time for the expression of the leukemic predisposition that characterizes the natural history of these disorders. However, other factors related to G-CSF may also be operative in the setting of congenital neutropenia. (Blood. 2000;96:429-436)

Random locomotion and chemotaxis of human blood polymorphonuclear leukocytes from a patient with leukocyte adhesion deficiency-1: normal displacement in close quarters via chimneying.

The beta2 integrins are known to be important in the motile function of leukocytes in general and in the adhesive response to inflammatory stimuli in particular. In the current study, under direct microscopic observation with concomitant time-lapse video recording, we examined the locomotion of human blood PMN from a patient with Leukocyte Adhesion Deficiency-1 (LAD), a disorder in which beta2 integrins on the cell surface are markedly deficient in number or function. In thin slide preparations such that the leukocytes were somewhat compressed between slide and cover slip, PMNLAD exhibited normal random locomotion and chemotaxis, apparently by using the opposing surfaces to generate the force for locomotion (chimneying). In thicker preparations, an adherence deficit was evident, but chemotaxis still occurred, even by PMNLAD anticoagulated in EDTA. Consistent with the paucity of beta2 integrins on the surface of the PMNLAD was their failure to aggregate in the presence of antibodies to beta2 integrins, even when they had been brought together by chemotaxis. We relate these findings to the reported independence from integrins of PMN in the lung vasculature in LAD, as well as in certain experimental conditions.

Regulation of polymorphonuclear leukocyte phagocytosis by myosin light chain kinase after activation of mitogen-activated protein kinase.

Polymorphonuclear leukocyte (PMNL) phagocytosis mediated by FcgammaRII proceeds in concert with activation of the mitogen-activated protein (MAP) kinase, extracellular signal-regulated kinase ERK2. We hypothesized that myosin light chain kinase (MLCK) could be phosphorylated and activated by ERK, thereby linking the MAP kinase pathway to the activation of cytoskeletal components required for pseudopod formation. To explore this potential linkage, PMNLs were challenged with antibody-coated erythrocytes (EIgG). Peak MLCK activity, 3-fold increased over controls, occurred at 4 to 6 minutes, corresponding with the peak rate of target ingestion and ERK2 activity. The MLCK inhibitor ML-7 (10 micromol/L) inhibited both phagocytosis and MLCK activity to basal values, thereby providing further support for the linkage between the functional response and the requirement for MLCK activation. The MAPK kinase (MEK) inhibitor PD098059 inhibited phagocytosis, MLCK activity, and ERK2 activity by 80% to 90%. To directly link ERK activation to MLCK activation, ERK2 was immunoprecipitated from PMNLs after EIgG ingestion. The isolated ERK2 was incubated with PMNL cytosol as a source of unactivated MLCK and with MLCK substrate; under these conditions ERK2 activated MLCK, resulting in phosphorylation of the MLCK substrate or of the myosin light chain itself. Because MLCK activates myosin, we evaluated the effect of directly inhibiting myosin adenosine triphosphatase using 2,3-butanedione monoxime (BDM) and found that phagocytosis was inhibited by more than 90% but MLCK activity remained unaffected. These results are consistent with the interpretation that MEK activates ERK, ERK2 then activates MLCK, and MLCK activates myosin. MLCK activation is a critical step in the cytoskeletal changes resulting in pseudopod formation.

Actin cytoskeletal function is spared, but apoptosis is increased, in WAS patient hematopoietic cells.

Mutations in the Wiskott-Aldrich syndrome protein (WASP) have been hypothesized to cause defective actin cytoskeletal function. This resultant dysfunction of the actin cytoskeleton has been implicated in the pathogenesis of Wiskott-Aldrich syndrome (WAS). In contrast, it was found that stimulated actin polymerization is kinetically normal in the hematopoietic lineages affected in WAS. It was also found that the actin cytoskeleton in WAS platelets is capable of producing the hallmark cytoarchitectural features associated with activation. Further analysis revealed accelerated cell death in WAS lymphocytes as evidenced by increased caspase-3 activity. This increased activity resulted in accelerated apoptosis of these cells. CD95 expression was also increased in these cells, suggesting an up-regulation in the FAS pathway in WAS lymphocytes. Additionally, inhibition of actin polymerization in lymphocytes using cytochalasin B did not accelerate apoptosis in these cells. This suggests that the accelerated apoptosis observed in WAS lymphocytes was not secondary to an underlying defect in actin polymerization caused by mutation of the WAS gene. These data indicate that WASP does not play a universal role in signaling actin polymerization, but does play a role in delaying cell death. Therefore, the principal consequence of mutations in the WAS gene is to accelerate lymphocyte apoptosis, potentially through up-regulation of the FAS-mediated cell death pathway. This accelerated apoptosis may ultimately give rise to the clinical manifestations observed in WAS. (Blood. 2000;95:1283-1292)

Syk activation initiates downstream signaling events during human polymorphonuclear leukocyte phagocytosis.

We investigated the requirement for Syk activation to initiate downstream signaling events during polymorphonuclear leukocyte (PMN) phagocytosis of Ab-coated erythrocytes (EIgG). When PMN were challenged with EIgG, Syk phosphorylation increased in a time-dependent manner, paralleling the response of PMN phagocytosis. Pretreatment of PMN with piceatannol, a Syk-selective inhibitor, blocked EIgG phagocytosis and Syk phosphorylation. We found that piceatannol inhibited protein kinase Cdelta (PKCdelta) and Raf-1 translocation from cytosol to plasma membrane by >90%. Extracellular signal-regulated protein kinase-1 and -2 (ERK1 and ERK2) phosphorylation was similarly blocked. We also investigated phosphatidylinositide 3-kinase (PI 3-kinase) activity and Syk phosphorylation using piceatannol, wortmannin, and LY294002, inhibitors of PI 3-kinase. The phosphorylation of Syk preceded the activation of PI 3-kinase. Both wortmannin and piceatannol inhibited PI 3-kinase, but only piceatannol inhibited Syk. In contrast to piceatannol, wortmannin did not inhibit PKCdelta and Raf-1 translocation. To elucidate signaling downstream of Syk activation, we assessed whether the cell-permeable diacylglycerol analogue didecanoylglycerol could normalize PMN phagocytosis, PKCdelta and Raf-1 translocation, and ERK1 and ERK2 phosphorylation inhibited by piceatannol. The addition of didecanoylglycerol to the Syk-inhibited phagocytosing PMN normalized all three without a concomitant effect on PI 3-kinase activity and Syk phosphorylation. We conclude that Syk activation following Fcgamma receptor engagement initiates downstream signaling events leading to mitogen-activated protein kinase activation independent of PI 3-kinase activation.

Identification of the molecular genetic defect of patients with methemoglobin M-Kankakee (M-Iwate), alpha87 (F8) His --> Tyr: evidence for an electrostatic model of alphaM hemoglobin assembly.

We determined that the molecular defect of 2 patients with hemoglobin (Hb) M-Kankakee [Hb M-Iwate, alpha87 (F8) His --> Tyr] resides in the alpha1-globin gene. The proportion of Hb M observed is higher than that predicted for an alpha1-globin variant. Our evidence suggests that the greater-than-expected proportion of Hb M-Kankakee results from preferential association of the electronegative beta-globin chains with the alpha(M)-globin chains that are more electropositive than normal alpha-globin chains.

Sphingosine blocks human polymorphonuclear leukocyte phagocytosis through inhibition of mitogen-activated protein kinase activation.

In the present study, we investigated the mechanism by which sphingosine and its analogues, dihydrosphingosine and phytosphingosine, inhibit polymorphonuclear leukocyte (PMN) phagocytosis of IgG-opsonized erythrocytes (EIgG) and inhibit ERK1 and ERK2 phosphorylation. We used antibodies that recognized the phosphorylated forms of ERK1 (p44) and ERK2 (p42) (extracellular signal-regulated protein kinases 1 and 2). Sphingoid bases inhibited ERK1 and ERK2 activation and phagocytosis of EIgG in a concentration-dependent manner. Incubation with glycine, N,N'-[1, 2-ethanediylbis(oxy-2, 1-phenylene)]bis[N-[2-[(acetyloxy)methoxy]-2-oxoethyl]]-bis[ (acetylox y)methyl]ester (BAPTA,AM), an intracellular chelator of calcium, failed to block either phagocytosis or ERK1 and ERK2 phosphorylation, consistent with the absence of a role for a calcium-dependent protein kinase C (PKC) in ERK1 and ERK2 phosphorylations. Western blotting demonstrated that sphingosine inhibited the translocation of Raf-1 and PKCdelta from PMN cytosol to the plasma membrane during phagocytosis. These data are consistent with the interpretation that sphingosine regulates ERK1 and ERK2 phosphorylation through inhibition of PKCdelta, and this in turn leads to inhibition of Raf-1 translocation to the plasma membrane. Consistent with this interpretation, the sphingosine-mediated inhibition of phagocytosis, ERK2 activation, and PKCdelta translocation to the plasma membrane could be abrogated with a cell-permeable diacylglycerol analog. The increase in the diacylglycerol mass correlated with the translocation of PKCdelta and Raf-1 to the plasma membrane by 3 minutes after the initiation of phagocytosis. Additionally, the diacylglycerol analog enhanced phagocytosis by initiating activation of PKCdelta and its translocation to the plasma membrane. Because PMN generate sufficient levels of sphingosine by 30 minutes during phagocytosis of EIgG to inhibit phagocytosis, it appears that sphingosine can serve as an endogenous regulator of EIgG-mediated phagocytosis by downregulating ERK activation.

The formation of ceramide-1-phosphate during neutrophil phagocytosis and its role in liposome fusion.

Ceramide, a product of agonist-stimulated sphingomyelinase activation, is known to be generated during the phagocytosis of antibody-coated erythrocytes by polymorphonuclear leukocytes. Agonist-stimulated formation of ceramide-1-phosphate is now shown to occur in 32PO4-labeled neutrophils. Ceramide-1-phosphate is formed by a calcium-dependent ceramide kinase, found predominately in the neutrophil plasma membrane. The neutrophil kinase is specific for ceramide because, in contrast to the bacterial diglyceride kinase, ceramide is not phosphorylated under conditions specific for diglyceride phosphorylation. Conversely, 1,2-diacylglycerol does not serve as substrate for the neutrophil ceramide kinase. Ceramide kinase activation occurs in a time-dependent fashion, reaching peak activity 10 min after formyl peptide stimulation and challenge with antibody-coated erythrocytes. The lipid kinase activity is optimal at pH 6.8. Because the formation of the phagolysosome is a critical event in phagocytosis, the effect of ceramide-1-phosphate in promoting the fusion of liposomes was determined. Both the addition of increasing concentrations of sphingomyelinase D and ceramide-1-phosphate promoted liposomal fusion. In summary, ceramide-1-phosphate is formed during phagocytosis through activation of ceramide kinase. Ceramide-1-phosphate may promote phagolysosome formation.

Activation of a plasma membrane-associated neutral sphingomyelinase and concomitant ceramide accumulation during IgG-dependent phagocytosis in human polymorphonuclear leukocytes.

The sphingomyelin cycle, which plays an important role in regulation of cell growth, differentiation, and apoptosis, involves the formation of ceramide by the action of a membrane-associated, Mg2+-dependent, neutral sphingomyelinase and/or a lysosomal acid sphingomyelinase. In human polymorphonuclear leukocytes (PMNs), ceramide production correlates with and plays a role in the regulation of functional responses such as oxidant release and Fcgamma receptor-mediated phagocytosis. To increase our understanding of the sphingomyelin cycle in human PMNs, the cellular location of neutral and acid sphingomyelinases was investigated in resting, formylmethionylleucylphenylalanine (FMLP)-activated, and FMLP-activated PMNs engaged in phagocytosis. In resting PMNs, a Mg2+-dependent, neutral sphingomyelinase was the predominant activity and was localized to the plasma membrane fractions along with the majority of ceramide. Upon FMLP-activation, there was a 1. 9-fold increase in this neutral, Mg2+-dependent sphingomyelinase activity, which increased to 2.7-fold subsequent to phagocytosis of IgG opsonized targets. This increase in sphingomyelinase activity was restricted to the plasma membrane fractions, which were also the site of increased ceramide levels. Phospholipase D (PLD) activity, which is a target of ceramide action and is required for phagocytosis, was also found primarily in the plasma membrane fractions of FMLP-activated and phagocytosing PMNs. Our findings indicate that in human PMNs engaged in phagocytosis, the sphingomyelin cycle is restricted to the plasma membrane where intracellular targets of ceramide action, such as PLD, are localized.

Oscillatory pericellular proteolysis and oxidant deposition during neutrophil locomotion.

To better understand the mechanism of leukocyte migration in complex environments, model extracellular matrices were prepared using gelatin, Hanks' solution, Bodipy-BSA (fluorescent upon proteolysis), and dihydrotetramethylrosamine or hydroethidine (fluorescent upon oxidation). Using quantitative microfluorometry, neutrophil-mediated extracellular pulses of reactive oxygen metabolites (ROMs) and pericellular proteolysis were periodically observed showing that these functions occur as quantal bursts. However, chronic granulomatous disease neutrophils, which do not produce ROMs, did not display ROM deposition. Matrices show an alternating pattern of green (proteolytic) and red (oxidative) fluorescence, indicating these functions are out of phase. Electric fields phase-matched with metabolic oscillations, which increase the amplitude of intracellular NAD(P)H oscillations, increase ROM deposition and pericellular proteolysis; this further supports the link between intracellular chemical oscillators and extracellular functions. This phase relationship may allow ROMs to inactivate protease inhibitors, followed by protease activation.

Severe chronic neutropenia: pathophysiology and therapy.

The development of recombinant-met human granulocyte-colony stimulating factor (r-metHuG-CSF) for clinical use has had a major influence on the treatment of many diseases. This impact has perhaps been greatest for treatment of severe chronic neutropenia (SCN) conditions for which there were no predictably effective treatment before the availability of these growth factors, particularly r-metHuG-CSF (Filgrastim, Amgen Inc, Thousand Oaks, CA; or Lenograstim, Rhone-Poulenc Rorer, Milan, Italy). Based on careful studies in many countries it is now known that more than 95% of these patients will respond promptly to r-metHuG-CSF treatment with normalization of the blood neutrophil levels and reduction in the occurrence of both major and minor consequences of their severe neutropenia. The availability of this treatment will undoubtedly lead to much additional research on the mechanisms governing neutrophil production and the basic mechanisms that can cause neutropenia among patients who have SCN. Among patients who have SCN those who are diagnosed to have severe congenital neutropenia (Kostmann's syndrome) or Shwachman-Diamond syndrome are at risk of developing myelodysplasia and/or acute myelogenous leukemia. The role of r-metHuG-CSF in facilitating the risk remains to be determined. Thus, it is important that long-term evaluation of the safety and efficacy of treatment of SCN and cooperation in research on these rare conditions proceed under the auspices of an international registry monitoring the clinical outcome of patients with severe congenital neutropenia.

Mitogen-activated protein kinase activation during IgG-dependent phagocytosis in human neutrophils: inhibition by ceramide.

In FMLP-activated polymorphonuclear leukocytes (PMNs) challenged with IgG-opsonized erythrocytes (EIgG), the termination of phagocytosis correlates with an accumulation of ceramide, a product of sphingolipid metabolism. Furthermore, the exogenous addition of short chain ceramides inhibits EIgG-mediated phagocytosis. In the present study, we identified p42 and p44 mitogen-actived protein (MAP) kinases, referred to as extracellular signal-regulated kinases ERK2 and ERK1, respectively, as intracellular targets of ceramide action during Fc gammaR-mediated phagocytosis. The tyrosine phosphorylation of ERK1 and ERK2 increased within 30 s of addition of EIgG, with maximal phosphorylation by 1 to 5 min. By 30 min, ERK1 and ERK2 were almost completely dephosphorylated. The kinetics of ERK1 and ERK2 tyrosine phosphorylation indicated that MAP kinase activation preceded target ingestion. N-Acetylsphingosine (C2-ceramide) inhibited phagocytosis, reduced ERK1 and ERK2 phosphorylation to basal levels, and reduced ERK1 and ERK2 activity by 85 to 90% and 70 to 80%, respectively. In contrast, N-acetyldihydrosphingosine (dihydro-C2-ceramide) had no effect on either tyrosine phosphorylation or activity of ERK1 and ERK2. In the presence of the MAP kinase kinase (MEK) inhibitor, PD 098059, phagocytosis was reduced by approximately 50%, while ERK1 and ERK2 activity was reduced by 85 to 90%. Thus, engagement of Fc gammaRs led to ERK1 and ERK2 phosphorylation and activation, and the activation of these enzymes was critical for phagocytosis. Furthermore, the inhibition of phagocytosis by C2-ceramide correlated with the inhibition of tyrosine phosphorylation and activation of ERK1 and ERK2. These results suggest that ceramides generated during phagocytosis act on the MAP kinase signaling pathway, ultimately "turning off" the phagocytic response.

Ceramide inhibits IgG-dependent phagocytosis in human polymorphonuclear leukocytes.

Ceramide is a product of agonist-induced sphingolipid metabolism in several cell types, including polymorphonuclear leukocytes (PMNs). In adherent PMNs, the kinetics of ceramide production correspond with the termination of fMLP-stimulated H2O2 release. Furthermore, short chain ceramides inhibit fMLP-mediated H2O2 release in adherent PMNs. In the present study, we investigated the effects of short chain ceramides and sphingoid bases on phagocytosis of IgG-opsonized erythrocytes (EIgG) by suspended PMNs activated with fMLP. N-Acetylsphingosine, N-acetylphytosphingosine, phytosphingosine, sphingosine, and dihydrosphingosine, but not N-acetyldihydrosphingosine, inhibited phagocytosis of EIgG. In contrast, these same lipids did not inhibit fMLP-mediated chemotaxis. Endogenous ceramide levels increased within the first few minutes of phagocytosis, with a significant (P < .05) accumulation by 30 minutes, the time by which phagocytosis was terminated. Neutral sphingomyelinase activity paralleled the increase in ceramide, consistent with the generation of ceramide by the hydrolysis of sphingomyelin. The N-acetyl-conjugated sphingols (C2 ceramides) blocked phosphatidylethanol formation indicating that phospholipase D (PLD) is an intracellular target of ceramide action. These data suggest that ceramides, generated through activation of the sphingomyelin cycle, act as negative regulators of Fc(gamma)R-mediated phagocytosis.