Staphylococcus aureus-stimulated mononuclear leucocyte-conditioned medium increases the neutrophil bactericidal activity, and augments oxygen radical production and degranulation in response to the bacteria.

Conditioned medium produced by human mononuclear leucocytes (MNL) stimulated with formalin-fixed, heat-killed Staphylococcus aureus enhanced the killing of opsonized S. aureus by human neutrophils. This enhancement was not seen when conditioned medium from non-stimulated MNL or medium cultured in the absence of both bacteria and MNL was used. There was a five-fold decrease in survival of bacteria when neutrophils were treated with conditioned medium. Conditioned medium-treated neutrophils showed increased initial rate of killing but after 20-30 min the rate of killing was similar to that of non-conditioned medium-treated leucocytes. The neutrophils treated with conditioned medium showed increased production of chemiluminescence, superoxide and lysosomal enzyme release (from both azurophilic and specific granules) in response to opsonized S. aureus. The results demonstrate that bacterial interaction with MNL leads to the release of mediators (cytokines) which potentiate the anti-bacterial function of neutrophils.     

Conditioned medium from stimulated mononuclear leukocytes augments human neutrophil-mediated killing of a virulent Acanthamoeba sp.

Human neutrophils in the presence of serum containing anti-amoeba antibody either lacked amoebicidal activity or were poorly amoebicidal for Acanthamoeba culbertsoni. In contrast, neutrophils preexposed for 1 h to supernatants from human peripheral blood mononuclear leukocytes (MNLs) stimulated with phytohemagglutinin demonstrated significant amoeba killing in the presence of serum containing anti-acanthamoeba antibodies. Supernatant from MNL cultured in the absence of phytohemagglutinin were not effective in stimulating significant activity in the neutrophils. Serum containing antibody promoted the adherence of many neutrophils to one amoeba. There was no significant difference between the ability of neutrophils treated with supernatants from stimulated MNLs (stimulated conditioned medium [sCM]) and supernatants from nonstimulated MNLs (nonstimulated conditioned medium [nsCM]) in their binding to acanthamoeba. The effects of sCM on neutrophils was a general phenomenon. For example, the sCM but not the nsCM enhanced the antibody-dependent neutrophil-mediated cytotoxicity against three tumor targets (K562 erythroid myeloid leukemia cell line, B16 melanoma, and P815 (DBA/2 mastocytoma). Furthermore, the sCM but not the nsCM increased the bactericidal (against Staphylococcus aureus and Streptococcus pneumoniae) and fungicidal (against Torulopsis glabrata) activity of the neutrophil. The sCM but not the nsCM contained activities which inhibited neutrophil migration and stimulated a respiratory burst in these leukocytes. These results suggest that the neutrophil antimicrobial power can be increased by exposing the leukocytes to MNL mediators.     

Differential effects of small tumour necrosis factor-alpha peptides on tumour cell cytotoxicity, neutrophil activation and endothelial cell procoagulant activity.

Tumour necrosis factor-alpha (TNF-alpha) is a pluripotent cytokine with its receptors distributed throughout many different cell types. Because of the diverse effects of the cytokine, it is difficult to clearly define its role in infection and immunity, and appreciate its clinical therapeutic value. We have identified peptides derived from the primary amino acid sequence of human TNF-alpha that have neutrophil-stimulating activity, as measured by enhanced chemiluminescence and superoxide production, and peptides which are both directly cytotoxic for tumour cells (WEHI-164) in vitro and also prevent TNF binding to tumour cells. However, only one of these neutrophil-stimulating peptides was toxic for tumour cells in vitro. Our results indicate that the region of amino acids 54-94 of human TNF-alpha has previously undescribed human neutrophil-stimulatory activity, while peptides encompassing the regions 43-68 and 132-150, which are in close proximity, as indicated in the recently determined three-dimensional structure of human TNF-alpha, have in vitro anti-tumour activity. These peptides also slowed tumour growth or induced tumour regression in WEHI-164 tumour-bearing mice. The peptide 73-94, which activated neutrophils but which was not cytotoxic for tumour cells in vitro, also caused in vivo tumour regression, presumably by activating neutrophils with the consequent release of free radicals at the tumour site. Peptide 63-83, which was able to activate neutrophils in vitro, did not possess tumour regression activity in vivo. The TNF peptides described in this report did not elicit procoagulant activity in cultured bovine aortic endothelial cells and as such are devoid of at least one of the potentially lethal side-effects of elevated TNF levels in vivo.     

Tetrandrine, a plant alkaloid, inhibits the production of tumour necrosis factor-alpha (cachectin) hy human monocytes.

Human mononuclear leucocytes (MNL) or the adherent fraction (monocytes) produced tumour necrosis factor-alpha (TNF-alpha) (by ELISA) in culture when stimulated with killed Staphylococcus aureus. The bisbenzylisoquinoline alkaloid, tetrandrine inhibited the capacity of MNL and monocytes to produce TNF-alpha at a concentration range of 0.1 to 5 micrograms/ml. Tetrandrine may be potentially useful in the treatment of inflammatory diseases in which TNF-alpha plays a major role.     

Human neutrophils require activation by mononuclear leucocyte conditioned medium to kill the pathogenic free-living amoeba, Naegleria fowleri

Naegleria fowleri is a free-living amoeba which causes a fulminant and rapidly fatal meningoencephalitis in man. Human neutrophils fail to kill the amoeba in vitro, but can do so if they are exposed to conditioned medium (CM) from PHA stimulated mononuclear leucocytes (MNLs). Specific antibody or complement was required to effect amoeba killing by CM modified neutrophils. Only short time exposure of the leucocytes to CM was required to endow them with amoebicidal properties. The CM was also shown to contain neutrophil migration inhibition activity and an activity(ies) which induced a respiratory burst in neutrophils. The results highlight the importance of MNL products other than specific antibody in neutrophil anti-microbial activity.     

Staphylococcus aureus and derived exotoxins induce nuclear factor kappa B-like activity in murine bone marrow macrophages.

Heat-killed gram-positive Staphylococcus aureus as well as S. aureus-derived exotoxins B and toxic shock syndrome toxin 1 can induce nuclear factor kappa B (NF-kappa B)-like activity in murine bone marrow macrophages. The induction of NF-kappa B-like activity in murine macrophages by S. aureus was as effective as induction by tumor necrosis factor alpha (TNF-alpha) or lipopolysaccharides (LPS) and was observed in macrophages derived from LPS-sensitive and LPS-resistant mice. Stimulation of macrophages with S. aureus but not with the exotoxins resulted in the accumulation of TNF-alpha in the culture medium. The induction of NF-kappa B-like activity by S. aureus, however, clearly preceded TNF-alpha secretion and was not inhibited by a neutralizing serum against TNF-alpha. In addition, pretreatment of macrophages with the protein synthesis inhibitor cycloheximide or dexamethasone, which prevented the secretion of TNF-alpha from macrophages, did not interfere with the induction of NF-kappa B-like activity by S. aureus. This findings reveal the existence of bacterial components other than LPS which can induce NF-kappa B-like activity in susceptible cells.     

Bactericidal action of eosinophils from normal human blood.

The ability of normal human eosinophils to ingest and kill Staphylococcus aureus and Escherichia coli was investigated and compared with the reactions shown by neutrophils from the same donors. The rate of phagocytosis of S. aureus by eosinophils was 50% of that shown by neutrophils. Unlike neutrophils, eosinophils were not able to kill ingested S. aureus at low bacterium/phagocyte ratios. The degree of S. aureus killing increased with increasing ratios, being equal to that of neutrophils when bacterium/phagocyte ratios of about 15 were used. This was probably due to a better triggering of the eosinophil oxidase system at high bacterium/phagocyte ratios. The early kinetics of the association of bacteria with eosinophils, the perforation of the bacterial envelope and the inactivation of bacterial proteins, was monitored in the ML-35 mutant strain of E. coli. The association of E. coli with eosinophils was 70% of that with neutrophils. Eosinophils had only 25% of the capacity of neutrophils to perforate the E. coli envelope. E. coli loses its colony-forming ability when the bacterial envelope has been perforated, indicating that eosinophils also kill E. coli more slowly than do neutrophils. This was confirmed with a plating assay for colony formation. The perforation of E. coli is independent of peroxidase-mediated reactions. Hence, the defective bactericidal action of eosinophils is probably not related to the differences between myeloperoxidase and eosinophil peroxidase. On the other hand, the inactivation of bacterial proteins is peroxidase dependent and was also seen to occur to a lesser extent in eosinophils compared with neutrophils. We conclude that eosinophils ingest E. coli but only slowly perforate (kill) these bacteria and barely inactivate the bacterial enzymes. In contrast, neutrophils quickly ingest and perforate (kill) E. coli and quickly inactivate the bacterial enzymes.     

Killing of Coccidioides immitis by human peripheral blood mononuclear cells.

The ability of human peripheral blood mononuclear cells (MNL) obtained from healthy donors to kill the fungus Coccidioides immitis was examined in vitro with an assay that uses a single fungal particle per well. MNL killed 25.0% +/- 3.5% of a coccidioidal arthroconidial target, compared with the 4.7% +/- 2.9% killed by polymorphonuclear leukocytes obtained from the same donors (P = 0.012). Arthroconidial killing by MNL was not dependent on donor delayed dermal hypersensitivity to spherulin. Killing of another fungal target, Candida glabrata, was not significantly different between MNL and polymorphonuclear leukocytes (P = 0.783). Depletion of monocytes from MNL with Sephadex G-10 resulted in a significant reduction in arthroconidial killing (21.4% +/- 13.6% versus 2.4% +/- 3.4%; P = 0.025), while enrichment of monocytes by Percoll density gradient centrifugation or plastic adherence resulted in significantly increased arthroconidial killing compared with that by MNL (P = 0.005 and 0.001, respectively). Killing of 96-h spherules by MNL was 7.3% +/- 3.1%, significantly less than the 21.4% +/- 2.8% killing of arthroconidia in the same experiments (P = 0.016). Incubation of MNL with human recombinant gamma interferon or tumor necrosis factor alpha did not result in increased MNL killing of coccidioidal arthroconidia under various conditions. These results suggest that MNL have an inherent ability to kill coccidioidal arthroconidia in vitro which is not dependent on prior host exposure to C. immitis. This activity appears to reside in peripheral blood monocytes.     

Role of the extracellular signal-regulated protein kinase cascade in human neutrophil killing of Staphylococcus aureus and Candida albicans and in migration

Killing of Staphylococcus aureus and Candida albicans by neutrophils involves adherence of the microorganisms, phagocytosis, and a collaborative action of oxygen reactive species and components of the granules. While a number of intracellular signalling pathways have been proposed to regulate neutrophil responses, the extent to which each pathway contributes to the killing of S. aureus and C. albicans has not been clearly defined. We have therefore examined the effect of blocking one such pathway, the extracellular signal-regulated protein kinase (ERK) cascade, using the specific inhibitor of the mitogen-activated protein kinase/ERK kinase, PD98059, on the ability of human neutrophils to kill S. aureus and C. albicans. Our data demonstrate the presence of ERK2 and a 43-kDa form of ERK but not ERK1 in human neutrophils. Upon stimulation with formyl methionyl leucyl phenylalanine (fMLP), the activities of both ERK2 and the 43-kDa form were stimulated. Despite abrogating the activity of both ERK forms, PD98059 only slightly reduced the ability of neutrophils to kill S. aureus or C. albicans. This is consistent with our finding that PD98059 had no effect on neutrophil adherence or degranulation, although pretreatment of neutrophils with PD98059 inhibited fMLP-stimulated superoxide production by 50%, suggesting that a change in superoxide production per se is not strictly correlated with microbicidal activity. However, fMLP-stimulated chemokinesis was markedly inhibited, while random migration and fMLP-stimulated chemotaxis were partially inhibited, by PD98059. These data demonstrate, for the first time, that the ERK cascade plays only a minor role in the microbicidal activity of neutrophils and that the ERK cascade is involved primarily in regulating neutrophil migration in response to fMLP.     

Localization of tumour necrosis factor production in cells at the materno/fetal interface in human pregnancy.

Biologically active tumour necrosis factor (TNF) was detected in medium conditioned by incubation with explants of human pregnancy decidua or fetal chorionic villous tissue, taken in the first trimester and at term. Addition of endotoxin increased TNF release in most cases. ELISA assays gave similar results for TNF-alpha and also demonstrated low levels of TNF-beta. Using cell populations purified by flow cytometry, secretion of biologically active TNF was shown to be localized to the macrophages. Cytotrophoblast purified from term amniochorion produced no TNF. Both decidual and chorionic villous tissue at term contained mRNA for TNF-alpha and TNF-beta. TNF-alpha mRNA was confined to decidual macrophages in first trimester tissue, and was not present in chorionic cytotrophoblast. TNF-beta mRNA, in contrast, was detected in both macrophage and non-macrophage populations in term decidua.     

An in vivo and in vitro study of selenium deficiency and infection in rats

Selenium deficiency in rats impairs the ability of neutrophils and peritoneal macrophages to kill Candida albicans organisms in vitro. In contrast, killing of Salmonella typhimurium and Staphylococcus aureus organisms is unaffected by the deficiency. Survival of rats after intraperitoneal injection of 8 X 10(7) S. aureus organisms was not affected by Se deficiency, but a 5-fold increase in the dose (4 X 10(8) S. aureus organisms) led to a significantly greater mortality in the Se deficient rats.     

Effect of a factor released by K562 malignant cells in culture on human neutrophil bactericidal activity.

We have previously demonstrated that k562 malignant cells in culture contain and release a low-molecular-mass (8-kDa) factor that inhibits adherence-related functions of neutrophils but does not alter fMet-Leu-Phe- or phorbol ester-induced oxidative burst (M. Amar, N. Amit, T. Pham Huu, S. Chollet-Martin, M.T. Labro, M.A. Gougerot-Pocidalo, and J. Hakim, J. Immunol. 144:4749-4756, 1990). In this study, we investigated the effects of this factor, referred to as inhibitory factor 1 (IF1), on the bactericidal activity of human polymorphonuclear cells (PMNs) on Staphylococcus aureus opsonized in various ways. S. aureus was used either nonopsonized or opsonized with heat-inactivated serum or normal serum containing complement factors. The bactericidal activity of PMNs preincubated with IF1-treated or control medium was examined by counting the surviving bacteria. The ability of IF1-treated PMNs to kill bacteria was diminished when they were opsonized with normal serum. When S. aureus was not opsonized or was opsonized with heat-inactivated serum, the bactericidal activity of IF1-treated PMNs was similar to that of controls. Likewise, the phagocytosis of IF1-treated PMNs was diminished when S. aureus was opsonized with normal serum but was not altered when S. aureus was not opsonized or was opsonized with heat-inactivated serum. These results suggest that the decrease in killing might be due to defective ingestion. The chemiluminescence response of IF1-treated PMNs was inhibited when S. aureus was not opsonized or was opsonized with normal serum. No effect on chemiluminescence was observed when S. aureus was opsonized with heat-inactivated serum. These results suggest that IF1 interferes not only with S. aureus stimulation of PMNs via complement receptors but also with oxygen-dependent bactericidal activity.     

Human tumour necrosis factor-alpha (TNF-alpha) directly stimulates arachidonic acid release in human neutrophils.

The ability of tumour necrosis factor-alpha (TNF-alpha) to directly stimulate phospholipid turnover from human neutrophils was studied. Stimulation with recombinant human (rH) TNF-alpha induced the release of significant amounts of radioactivity from [3H]arachidonic acid-labelled neutrophils. This stimulation was equipotent to that induced by the bacterial tripeptide formyl-methionyl-leucylphenylalanine (FMLP). The time of maximum stimulated release varied between donors, with the most common maximal stimulation being 45 min. Dose-response experiments indicated that 100-1000 U/ml rH TNF-alpha were required for the maximum stimulatory effect. High-performance liquid chromatography analysis of the supernatants revealed that the radioactivity was associated with arachidonic acid, but not with its metabolites, indicating that TNF-alpha stimulates the release of arachidonic acid from cellular phospholipids but does not stimulate its metabolism. A comparison of TNF-alpha with other cytokines indicated that stimulation of arachidonic acid release paralleled the 'priming' of neutrophils for enhanced superoxide production, raising the possibility that phospholipid turnover and priming of neutrophils are causally related.     

Inhibition of tumour necrosis factor alpha (TNF-alpha)-induced neutrophil respiratory burst by a TNF inhibitor.

Tumour necrosis factor alpha (TNF-alpha) plays an important role in microbial defence and tissue damage by activating neutrophils. Therefore the ability of natural molecules to regulate the activity of TNF-alpha is likely to be of major importance in our understanding of the mechanisms of inflammation. We have examined the effects of a highly purified urine-derived TNF inhibitor (TNF inh) on the TNF-alpha-induced respiratory burst in human neutrophils. TNF-alpha inh-treated TNF-alpha was unable to stimulate a neutrophil lucigenin-dependent chemiluminescence response and superoxide formation. Treatment of TNF with the inhibitor also significantly reduced the priming ability of TNF-alpha for a response to the peptide f-met-leu-phe. These results show that the ability of TNF-alpha to induce a key neutrophil response is amenable to regulation by the TNF-alpha inh.     

Inhibition of endothelial interleukin-8 production and neutrophil transmigration by Staphylococcus aureus beta-hemolysin

Neutrophils play a crucial role in the host response to infection with Staphylococcus aureus, which is a major human pathogen capable of causing life-threatening disease. Interleukin-8 (IL-8) is a potent chemoattractant and activator of neutrophils. We previously reported that S. aureus secretes a factor that suppresses IL-8 production by human endothelial cells. Here we isolated an inhibitor of IL-8 production from the supernatant and identified it as staphylococcal beta-hemolysin. Beta-hemolysin reduced IL-8 production without cytotoxicity to endothelial cells. Pretreatment with beta-hemolysin decreased the expression of both IL-8 mRNA and protein induced by tumor necrosis factor alpha (TNF-alpha). Migration of neutrophils across TNF-alpha-activated endothelium was also inhibited by beta-hemolysin. In contrast, beta-hemolysin had no effect on intercellular adhesive molecule 1 expression in activated endothelial cells. These results showed that beta-hemolysin produced by S. aureus interferes with inflammatory signaling in endothelial cells and may help S. aureus evade the host immune response.     

Tumour necrosis factor-alpha potentiates CR3-induced respiratory burst by activating p38 MAP kinase in human neutrophils

CR3 and Fc gamma Rs are the main receptors involved in the phagocytic process leading to engulfment and killing of microbes by production of reactive oxygen intermediates (ROI) and degranulation. Various inflammatory mediators, such as tumour necrosis factor-alpha (TNF-alpha) and lipopolysaccharide (LPS), are known to prime neutrophils leading to increased bactericidal responses, but the underlying mechanism of priming has only been partially elucidated. The purpose of this study was to investigate how TNF-alpha primes neutrophils for subsequent stimuli via either CR3 or Fc gamma R. The receptors were specifically activated with pansorbins (protein-A-positive Staphylococcus aureus) coated with anti-CR3, anti-Fc gamma RIIa, or anti-Fc gamma RIIIb monoclonal antibody. Activation of neutrophils with these particles resulted in ROI production as measured by chemiluminescence. Anti-CR3 pansorbins induced the most prominent ROI production in neutrophils. TNF-alpha potentiated the CR3-mediated respiratory burst but had little effect on that mediated by Fc gamma Rs. The priming effect of TNF-alpha on CR3-mediated ROI production is associated with an increased activation of p38 MAPK as well as tyrosine phosphorylation of p72(syk). Pretreatment of neutrophils with the inhibitors for p38 MAPK and p72(syk) markedly suppressed the respiratory burst induced by CR3. Furthermore, TNF-alpha induced about a three-fold increase in the expression of CR3 in neutrophils, an effect which is blocked by the p38 MAPK inhibitor. Taken together, these results showed that TNF-alpha potentiates the CR3-mediated respiratory burst in neutrophils not only by triggering a p38 MAPK-dependent up-regulation of CD11b/CD18 but also by modulating the signalling pathways.     

LIGHT enhances the bactericidal activity of human monocytes and neutrophils via HVEM

Human monocytes and neutrophils play major roles in clearing bacteria from human blood and tissues. We found that the herpes virus entry mediator (HVEM) was highly expressed in monocytes and neutrophils, and its interaction with "homologous to lymphotoxins, shows inducible expression, and competes with herpes simplex virus glycoprotein D for HVEM/tumor necrosis factor (TNF)-related 2" (LIGHT) enhanced bactericidal activity against Listeria monocytogenes and Staphylococcus aureus. The LIGHT-HVEM interaction increased levels of phagocytosis, interleukin (IL)-8, TNF-alpha, nitric oxide (NO), and reactive oxygen species (ROS) in monocytes and neutrophils. Anti-HVEM monoclonal antibody was able to block LIGHT-induced bactericidal activity, cytokine production (IL-8 and TNF-alpha), and ROS generation. Moreover, inhibition of ROS and NO production blocked LIGHT-induced bactericidal activity. Our results indicate that the LIGHT/HVEM interaction in monocytes and neutrophils contributes to antibacterial activity.     

Salmonellae activate tumor necrosis factor alpha production in a human promonocytic cell line via a released polypeptide.

Invasive strains of Salmonella spp. cause both systemic and localized infections in humans. The ability to resist infection and some aspects of the tissue pathology associated with the presence of Salmonella in the gastrointestinal tract have been shown to be mediated in part by the induction of tumor necrosis factor alpha (TNF-alpha), a proinflammatory cytokine produced by activated macrophages and lymphocytes. Recent reports indicate that TNF-alpha is involved in the induction of human immunodeficiency virus replication by Salmonella in the latently infected human promonocytic cell line U1. In the present study, we investigated the effects of Salmonella on TNF-alpha production in U1 cells and a related cell line, U38. Unlike Escherichia coli or Yersinia enterocolitica, salmonellae rapidly induce TNF-alpha expression in these cells through a released factor(s). Time course experiments show that the kinetics of TNF-alpha production by U38 cells stimulated with Salmonella conditioned medium closely resemble those observed in response to live Salmonella. The observation that TNF-alpha levels are elevated by 60 min after exposure to either bacteria or their conditioned medium suggests that the soluble inducer is continuously released or shed by the bacteria and that the signal acts rapidly to increase TNF-alpha production. Furthermore, the ability to produce the TNF-alpha inducer is shared by at least four Salmonella serotypes and does not correlate with the abilities to invade and to survive within phagocytes. Treatment of active conditioned medium with trypsin, but not low pH, high temperature, or urea, significantly inhibits its TNF-alpha-inducing effect on U38 cells, a finding which points to a polypeptide product of Salmonella as the mediator of TNF-alpha production. Gel filtration chromatography of Salmonella conditioned medium reveals two peaks of activity, consistent with molecular masses of approximately 150 and 110 kDa.     

Bacterial killing in vitro by abscess-derived neutrophils

In the absence of antimicrobial therapy, bacteria such as Bacteriodes fragilis, Escherichia coli and Proteus mirabilis may persist within an intra-abdominal abscess in the presence of large numbers of neutrophils which, under optimal conditions in vitro, can readily phagocytose and kill the same bacterial strains. Neutrophils taken from abscesses induced by gram-negative bacteria such as those above contain viable organisms. On incubation in vitro in the presence of serum, these neutrophils kill the bacteria phagocytosed in the abscess poorly, if at all, yet can readily kill organisms added in vitro. To determine possible mechanisms that might explain this, we examined the bactericidal activity in vitro of neutrophils from a range of abscesses induced by one or two species of bacteria plus an abscess-potentiating agent, bran. The organisms studied were B. fragilis, E. coli, P. mirabilis and Staphylococcus aureus. The killing in vitro of E. coli and P. mirabilis, engulfed within an abscess, was significantly less than that of the same organisms when they were added to the in-vitro assay. In contrast, the killing of S. aureus was similar, whether engulfed in vivo or in vitro. However, S. aureus was less susceptible to phagocytosis and killing in vitro than P. mirabilis or E. coli, and the killing of S. aureus during in-vitro incubation of neutrophils that had engulfed the organism with in the abscess was similar to that of the gram-negative bacteria engulfed within the abscess.(ABSTRACT TRUNCATED AT 250 WORDS)     

Eosinophil chemotactic factor release from neutrophils induced by stimulation with Schistosoma japonicum eggs

 The neutrophil is one of the sources of eosinophil chemotactic factor (ECF) in the presence of some stimulants. In the present study we showed that guinea-pig neutrophils could release ECF upon stimulation with Schistosoma japonicum eggs. ECF release from neutrophils began as early as 5 min after the stimulation and reached a peak at 20 min. When homogenate of the eggs was separated into a water-soluble fraction as soluble egg antigen (SEA) and a water-insoluble fraction (eggshell), both preparations possessed a potent neutrophil-stimulating activity to release ECF. The ECF release was dependent on the concentration of eggshells or SEA or on the number of neutrophils. The neutrophil-stimulating activity of eggshells was stable to heat, HCl, or pronase treatment but sensitive to NaOH treatment. When the eggs or eggshells were washed with acetone or Tween-20, they lost the neutrophil-stimulating activity to release ECF, indicating that the neutrophil-stimulating factor (NSF) possesses a lipid nature. The molecular weight of NSF extracted from the eggshells was estimated to be about 1000 Da by gel chromatography on Sephadex G25. The possible role of eggshells in the formation of eosinophil-rich granulomatous lesions in schistosomiasis japonica is discussed. Received: 13 May 1996 / Accepted: 25 July 1996