Enhanced inhibition of in vitro multiplication of Plasmodium falciparum by stimulated human polymorphonuclear leucocytes

The effect of normal human peripheral blood polymorphonuclear leucocytes on in vitro multiplication of Plasmodium falciparum malaria parasites was investigated. It was shown that normal neutrophils were able to phagocytose parasitized erythrocytes and free parasites and thus inhibit in vitro multiplication of the parasite. Stimulation of the neutrophils by phorbol myristate acetate, a potent stimulus of leucocyte oxidative metabolism, resulted in enhanced inhibition of parasite growth. Superoxide dismutase, scavenger of superoxide anion, catalase, inhibitor of hydrogen peroxide, and sodium azide, inhibitor of myeloperoxidase, did not abrogate the inhibitory ability of the neutrophils. The results indicate that polymorphonuclear leucocytes play an important role in the defence against P. falciparum malaria.     

Independence of complement on in vitro immune phagocytosis of plasmodium falciparum parasitised erythrocytes by human monocytes and polymorphonuclear leukocytes

Monocytes and polymorphonuclear leukocytes from normal blood donors phagocytosed preferentially Plasmodium falciparum-infected red blood cells (IRBC) in presence of sera from individuals living in areas endemic for malaria. Total complement or factor B heat inactivation of immune or normal serum does not alter opsonic activity directed against IRBC.     

Inhibition by lipid A-specific monoclonal antibodies of priming of human polymorphonuclear leucocytes by endotoxin

Exposure to lipopolysaccharide (LPS) primes polymorphonuclear leucocytes (PMNL) for enhanced release of oxygen metabolites after subsequent stimulation. The metabolic response of human PMNL primed with LPS and stimulated with formyl-methionyl-leucyl-phenylalanine (FMLP) was measured by chemiluminescence (CL) as a parameter for endotoxic activity. Polymyxin B (PMB) and monoclonal antibodies (MAbs) with specificity for lipid A were tested for inhibition of the priming effect of Re LPS of Salmonella minnesota R595, Rc LPS of Escherichia coli J5 and smooth LPS of E. coli O111. The CL response of PMNL primed with Re LPS or Rc LPS was higher than that of PMNL primed with smooth LPS. Pre-incubation of rough or smooth LPS with PMB caused dose-dependent inhibition of priming of PMNL. Two IgM MAbs, 8-2 and 26-20, which recognise different epitopes on the hydrophobic part of lipid A, also completely prevented priming of PMNL by either rough or smooth LPS. The dose-dependent inhibitory effect of both MAbs was similar to the inhibition by PMB. These results indicate that the binding of MAbs to the hydrophobic part of lipid A is important in blocking lipid A-mediated effects.     

Opsonic requirements and interaction of Haemophilus influenzae with human polymorphonuclear neutrophil leucocytes studied by luminol-enhanced chemiluminescence

We have used human polymorphonuclear leucocyte (PMNL)-dependent chemiluminescence (CL) to study bacteria opsonised with those factors in serum which are reported to be important in opsonisation of H. influenzae, and to determine whether alteration of various surface characteristics of H. influenzae influence those CL responses by PMNL. Although complement plays a role, immunoglobulin and a heat-labile factor(s) were found to be the principle stimulants of PMNL-dependent CL when H. influenzae was opsonised in pooled normal human serum. Acquisition of capsule (serotype a,b,c,e, or f) by an uncapsulated strain significantly (P less than 0.001) reduced its ability to stimulate PMNL-dependent CL, but the type of capsule did not discriminate between the strains in this regard. Surface-adherent capsule inhibited PMNL-dependent CL stimulation more than capsular material released into the supernatant. Altering the lipooligosaccharide composition of the bacterial cell wall also affected PMNL-dependent CL stimulation independent of capsule. We conclude that, although surface characteristics of H. influenzae influenced its ability to stimulate PMNL-dependent CL, these experiments provide no evidence to support the hypothesis that the increased virulence of serotype b capsulated strains compared with other capsulated types could be explained by any specific ability to avoid opsonisation.     

Phagocytosis and ingestion of influenza virus by human polymorphonuclear leucocytes in vitro: electronmicroscopy studies

Interaction of human polymorphonuclear leucocytes (PMNL) and influenza virus (IFV) was studied in vitro. At 0 degree C, the viral particles were bound extensively to the surface of the PMNL membrane with a ratio of about 1000 virus particles to a single PMNL. The binding was sensitive to neuraminidase, suggesting attachment through sialo-compound receptors. At 37 degrees C, the virus particles disappeared rapidly from the cell surface, about half of them being eluted and the remainder being endocytosed into the cytoplasmic vesicles. Immuno-gold electronmicroscopy suggests that the virus particles are ingested into phagosomal vesicles and lysed.     

Mechanisms of phagocytosis in human polymorphonuclear leucocytes

Human polymorphs have been found to ingest a wide variety of particles without the aid of serum, indicating that the cells possess a serum-independent mechanism of phagocytosis. Polymorphs have also been shown to possess a different and complementary mechanism of phagocytosis which depends on the presence of serum and serum components in the medium.

Ingestion of starch particles by human polymorphs in the absence of serum was unaffected by media at the extremes of physiological pH and at tonicities between 205 and 348 m-osmoles/l and by the presence in media of neutral and acid mucopolysaccharides.

Phagocytosis of starch particles was reversibly inhibited by media of high tonicity and irreversibly inhibited by low concentrations of bacterial lipopolysaccharide, which had a lethal effect on the cells. Serum was unable to promote phagocytosis by polymorphs in media of high tonicity but both untreated serum and inactivated serum promoted phagocytosis in media containing endotoxin, probably by neutralizing the action of the lipopolysaccharide.

Phagocytosis of starch particles by human polymorphs in the absence of serum was inhibited by prior treatment of the cells with iodoacetate, suggesting that serum-independent phagocytosis relies on glycolytic energy. Ingestion of starch particles by polymorphs, treated with iodoacetate, was largely contingent on the presence of serum in the suspending medium, but inactivated serum and individual plasma proteins also had a limited ability to promote phagocytosis. These results suggested that cell glycolysis is not the principal source of energy in the serum-dependent mechanism of phagocytosis.

Using hydrocarbon test particles, it was shown that combinations of either of the heat-labile components of complement (C′1 or C′2) with the C′4 component were active in promoting phagocytosis. Evidence was also presented that the C′3 component had some activity and another serum factor, which was only present in the heat-inactivated sera of some individuals, also had a limited ability to promote phagocytosis.

     

Antitoxin activity of human polymorphonuclear leucocytes

Human polymorphonuclear leucocytes (PMNL) inactivate Clostridium difficile cytotoxin and C. perfringens phospholipase C, but not C. perfringens enterotoxin. Both whole cells and sonicated suspensions possess activity, but mononuclear cell fractions of peripheral blood do not. Antitoxin activity closely correlates with cell concentration. The highest cell concentrations tested completely inactivated C. difficile cytotoxin by 2 min. Sucrose density gradient fractionation of PMNL showed antitoxin activity to be associated with myeloperoxidase, locating it in the primary or azurophil granules. Toxin inactivation was prevented by protease inhibitors suggesting that it is due to one of the neutral proteases present in these granules. PMNL are more active against C. difficile cytotoxin than purified chymotrypsin. PMNL may be a primary defence against certain bacterial exotoxins.     

Inhibition of human neutrophil degranulation by transforming growth factor-beta1

Neutrophils enter tissues including the uterus and are found in the endometrium in increased numbers prior to menses. In this environment, they are exposed to transforming growth factor (TGF)-beta1 produced by endometrial stromal and epithelial cells. We observed that incubation of neutrophils in vitro with TGF-beta1 at 1 pg/ml significantly reduced their secretion of lactoferrin in response to lipopolysaccharide (LPS). This effect was achieved with as little as 15 min of pretreatment with TGF-beta1. Inhibition of lactoferrin release by TGF-beta1 was observed irrespective of whether neutrophils were stimulated by ligands for Toll-like receptor (TLR)-2, TLR-4 or FPR, the G protein-coupled receptor for formylated peptides. Inhibition by TGF-beta1 was negated by SB-431542, a small molecule inhibitor that specifically blocks the kinase activity of the type I TGF-beta receptor (ALK5) In contrast to lactoferrin release, another important neutrophil function, interleukin (IL)-8 driven chemotaxis, was not affected by TGF-beta1 at 1 pg/ml or 100 pg/ml. We conclude that in tissues of the female reproductive tract, TGF-beta1 inhibition of neutrophil degranulation may prevent these cells from initiating an inflammatory response or releasing degradative enzymes that could potentially damage the oocyte or fetus.     

Inhibition of the growth of Plasmodium falciparum in vitro by covalent modification of hemoglobin

Antimalarial effects might be expected from compounds that modify hemoglobin. Dibromoaspirin and bis(dibromosalicyl) diesters decrease gelation of hemoglobin by specific covalent modification (acetylation and crosslinking) of this protein but do not interfere with oxygen transport. These compounds were toxic to malaria parasites when continuously present in culture, as were drugs with similar pharmacological effects such as indomethacin, ibuprofen, and phenylbutazone. Aspirin and acetaminophen were much less effective. When erythrocytes were pretreated with these compounds prior to parasite exposure, only dibromoaspirin and dibromosalicyl diesters prevented parasite development. The modified hemoglobin was highly resistant to digestion by cathepsin D and parasite proteases, suggesting that covalent modifications of hemoglobin that do not disrupt normal hemoglobin function have antimalarial effects.     

Enhancement of in vitro immunoglobulin synthesis of human lymphocytes by lysosomal enzymes from polymorphonuclear leucocytes.

The effect of human peripheral blood polymorphonuclear leucocyte (PMN) extracts and PMN granule lysates on in vitro immunoglobulin (Ig) synthesis by autologous peripheral blood mononuclear cells was studied. The mononuclear cells were cultured for 3 days with or without autologous plasma. Newly synthesized Ig in the culture supernatants was measured using 14C-labelled amino acids by an immune coprecipitation method. Upon addition of a PMN extract to plasma-free cultures Ig synthesis was stimulated, the mean stimulation index (SI) of cultures from thirteen individuals, including nine normals, three patients with rheumatoid arthritis and one with psoriatic arthritis being 1-8 +/- 0-2 in comparison with control cultures (P less than 0-05). By contrast, in 10% fresh autologous plasma, PMN extracts yielded a mean SI of 0-9 +/- 0-1 indicating inactivation of the active extracts by plasma inhibitors. In experiments using PMN granule lysates containing high concentrations of beta-glucuronidase and cultured in RPMI 1640, the mean stimulation index was 3-2 +/- 0-7. Stimulation of Ig synthesis was also produced by trypsin. Stimulation of Ig synthesis was also produced by trypsin. Stimulating factors in PMN extracts were inhibited by Trasylol, a protease inhibitor. These results indicate that trypsin and proteolytic lysosomal enzymes in PMN increase Ig synthesis of human peripheral blood mononuclear cells. They suggest a possible new role of PMN in the potentiation of immunoglobulin synthesis.     

Heparin inhibits FMLP and Con-A dependent activation of human polymorphonuclear leucocytes in vitro

The effects in vitro of heparin on different functions of human neutrophilic leucocytes were assessed. Granulocyte aggregation, enzyme release and superoxide anion generation induced by FMLP (10(-6) M), ConA (30 micrograms/ml), and A 23187 (5 microM) were evaluated. Heparin was able to inhibit, in a dose-dependent way, FMLP-mediated and ConA-mediated granulocyte activation. Heparin inhibited cellular aggregation at the final concentration of 100-500 micrograms/ml, while the inhibiting effect on enzyme release and superoxide anion production was present at lower concentrations (10-100 micrograms/ml). No activity was observed on A 23187-induced granulocyte stimulation. The inhibiting effect of heparin on FMLP-induced granulocyte activation proved to be time-dependent. The hypothesis that an interaction of heparin with cells may possibly initiate the inhibitory effect is proposed.     

Antitoxin activity of human polymorphonuclear leucocytes.

Human polymorphonuclear leucocytes (PMNL) inactivate Clostridium difficile cytotoxin and C. perfringens phospholipase C, but not C. perfringens enterotoxin. Both whole cells and sonicated suspensions possess activity, but mononuclear cell fractions of peripheral blood do not. Antitoxin activity closely correlates with cell concentration. The highest cell concentrations tested completely inactivated C. difficile cytotoxin by 2 min. Sucrose density gradient fractionation of PMNL showed antitoxin activity to be associated with myeloperoxidase, locating it in the primary or azurophil granules. Toxin inactivation was prevented by protease inhibitors suggesting that it is due to one of the neutral proteases present in these granules. PMNL are more active against C. difficile cytotoxin than purified chymotrypsin. PMNL may be a primary defence against certain bacterial exotoxins.     

Inhibition of human neutrophil chemotaxis in vitro by phenothiazines and related compounds.

Chlorpromazine (CPZ) and three other phenothiazines and the structurally related antidepressant drugs imipramine and amitriptyline were found to depress human neutrophil chemotactic responsiveness. A 7 X 10(-6)M solution of CPZ inhibited chemotaxis, whereas concentrations of the other tested drugs 10 to 1,000 times greater than this were needed to inhibit chemotaxis. This effect of CPZ could not, however, be demonstrated when testing neutrophils from patients treated with the drug. The inhibition of chemotaxis was reversible when CPZ-incubated neutrophils were washed before testing for chemotactic responsiveness. CPZ affects neutrophil funcjtion as well as other aspects of immune response.     

Chloroquine interaction with inflammatory human polymorphonuclear leucocytes

The molecularin vitro association of radiolabelled chloroquine (CQ) with both normal resting and inflammatory polymorphonuclear leucocytes (PMNs) was measured. For this purpose a suitable ligandassocation assay was developed to measure the cell association and the intracellular concentration of CQ. Under the influence of inflammatory stimuli PMNs display altered interaction with CQ. The intracellular concentration of CQ is reduced with 30 to 40% under inflammatory (disease) states when compared with non-inflammatory conditions. The mechanisms of CQ-PMN interaction associated with these altered intracellular concentrations of CQ are considered, with particular attention to the effects of rheumatic disease. Association experiments of CQ with PMNs performed in the presence of different established transport inhibitors showed that both diffusive uptake and carrier-mediated transport are involved in the cell accumulation of CQ in inflammatory PMNs. From these results, emphasis is given to three explanations for the decrease of the intracellular CQ concentration in inflamed PMNs:

1. the expansion of the PMN volume under inflammatory conditions;
2. the cytoplasmic or lysosomal pH changes and activation of the PMN Na⁺/H⁺ antiport by inflammatory stimuli; and
3. the exocytic release of the granules (degranulation).

Our data suggest that all these mechanisms, based on the events involved in inflammatory responses, may be involved in the decrease of the intracellular CQ concentration in inflammatory PMNs.     

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

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

Inhibition of phagosome-lysosome fusion in ovine polymorphonuclear leucocytes by Ehrlichia (Cytoecetes) phagocytophila

Ehrlichia (Cytoecetes) phagocytophila, the causative agent of tick-borne fever, is an intracellular bacterium that survives and multiplies within granulocytes and monocytes. In the present study, the possible fusion of lysosomes with phagosomes containing E. phagocytophila was investigated in poly-morphonuclear (PMN) cells of sheep infected with the agent, acid phosphatase cytochemistry and cationized ferritin being used as markers of primary and secondary lysosomal enzymes. Latex beads or Candida albicans were incubated with infected and uninfected PMN cells and labelled with the same lysosomal markers. Lysosomal enzymes labelled with the markers were commonly found in phagosomes containing latex beads or C. albicans, but there was no evidence of phagosome-lysosome (P-L) fusion in phagosomes containing E. phagocytophila. It was significant that in cells that contained E. phagocytophila, latex beads and C. albicans, P-L fusion occurred only in phagosomes containing latex beads or C. albicans. However, evidence of P-L fusion with phagosomes containing E. phagocytophila was obtained when PMN cells were incubated with oxytetracycline, which is known to inhibit synthesis of bacterial proteins. These findings indicate that E. phagocytophila is capable of inhibiting P-L fusion and that oxytetracycline depresses this capability.     

Ultrastructure of the polymorphonuclear leucocytes in human immunodeficiency virus infection

The ultrastructure of polymorphonuclear leucocytes (PMNL) was studied in 16 patients infected with the human immunodeficiency virus (HIV). PMNL were isolated from HIV-infected patients with CD4+ lymphocytes counts > 200/mm3 (without signs of active infection) (n = 12) (group 1), or < 200/mm3 (n = 4) (group 2), and from 16 healthy volunteers (group 3). Immunoelectron microscopy staining using an anti-beta 2 integrin antibody (anti-CD18) was performed on PMNL from three individuals of group 2 and of three individuals of group 3, before and after incubation with N-formyl-methionyl-leucylphenylalanine (f-MLP). The radical oxygen intermediates (ROI) production of PMNL was investigated by luminol-mediated chemiluminescence. A number of ultrastructural abnormalities in PMNL were found in a higher proportion in HIV-infected patients. These were: (a) an increase in the size of the Golgi apparatus and in the number of mitochondria, and in the quantity of endoplasmic reticulum; (b) some dysplastic features including large cytoplamic vacuoles, whorl of myelin, and nuclear pockets; (c) an increase prevalence of multivesicular bodies compared with control PMNL; (d) some cylindrical confronting cisternae and tubuloreticular structures. After anti-CD18 staining, gold particles were seen on the plasma membrane and more rarely inside the cytoplasm of PMNL from each group but no decrease in this staining was noted in HIV PMNL. Incubation with f-MLP similarly increased the immunostaining of the PMNL in each group. In vitro ROI production was significantly depressed for HIV PMNL compared with control PMNL. Some ultrastructural abnormalities observed in this study could support the possibility that one of the mechanisms underlying the qualitative functional defects of PMNL from HIV-infected patients may be related to some cytopathic effect.     

Enhancement of base hexose-monophosphate shunt activity of human polymorphonuclear leucocytes by human beta-interferon

The effect of human fibroblast interferon (HuIFN-beta) on the HMP shunt activity of human polymorphonuclear leucocytes (PMNLs) was examined. HuIFN-beta caused an increase in the base level of the HMP shunt activity. No significant increase was observed in the same PMNLs stimulated with opsonized zymosan. The augmentation of this property, generally associated with antimicrobial and cytotoxic properties of the PMNLs, may be of potential importance in host defences against microbial and malignant diseases.     

Lactoferrin-deficient neutrophil polymorphonuclear leucocytes in leukaemias: a semiquantitative and ultrastructural cytochemical study

Semiquantitative analysis of lactoferrin deficiency in neutrophil polymorphonuclear leucocytes in various haematological and non-haematological disease was carried out by scoring polymorphonuclear leucocytes stained for lactoferrin by the immunoperoxidase method. The staining patterns for lactoferrin were classified into four types (0-III) based on the intensity of reaction, and the sum of the ratings of 100 polymorphonuclear leucocytes was considered as "lactoferrin score" with a possible range of 0-300. As a result, significantly low lactoferrin-scores were frequently observed in acute leukaemias and the acute phase of chronic leukaemias. Of 35 cases with leukaemias, lactoferrin-negative polymorphonuclear leucocytes (type 0) were observed in the following cases: eight cases of acute myelogenous leukaemia (8/14), a case of chronic myelogenous leukaemia (1/10) in blast crisis, one of acute promyelocytic leukaemia (1/1), one of acute monocytic leukaemia (1/2), and a case of chronic myelomonocytic leukaemia (1/2) in a transitional phase to an acute myelomonocytic leukaemia. In two cases of acute myelogenous leukaemia, in which the majority of polymorphonuclear leucocytes were negative for lactoferrin, ultrastructural cytochemical study revealed total lack of specific granules in these polymorphonuclear leucocytes. This suggests that lactoferrin is localised in the specific granules of neutrophils as has been postulated previously by others.