Effect of two gold compounds on human polymorphonuclear leukocyte lysosomal function and phagocytosis

Lysosomal neutral proteases, once released, are considered to play an important role in the rheumatoid inflammatory process. The effect of two gold compounds on human polymorphonuclear lysosomal enzymes was studied during reverse endocytosis. Phagocytosis was assessed using dual-labeled liposomes. Auranofin, a new antirheumatic gold compound, reduces human polymorphonuclear leukocyte phagocytoses and lysosomal elastase and-glucuronidase release at a therapeutically achievable concentration (5 M), Sodium aurothiomalate was ineffective at this concentration.This study was aided by the South African Medical Research Council.     

Augmentation of human polymorphonuclear leukocyte adherence by interferon

Augmentation of human polymorphonuclear leucocyte adherence by alpha and gamma interferons occurred as early as 2 min after incubation. Enhancement of adherence occurred at optimal concentrations of 100-1,000 IU/ml. There was synergism between alpha and gamma interferons, but not between two subtypes of alpha interferon, on augmentation of adherence, indicating that alpha and gamma interferons act on different receptors on the polymorphonuclear leucocyte. Heat treatment at 65 degrees C for 30 min abolished the effect of interferon on adherence.     

Does tuftsin alter phagocytosis by human polymorphonuclear neutrophils?

The physiological significance of the putative phagocytosis-promoting peptide, tuftsin, was investigated by measurement of chemiluminescence generated during phagocytosis and by assay of the uptake of radiolabeled bacteria. We found no differences in either assay when we compared serum from splenectomized patients (which purportedly lacks tuftsin) with normal serum. Further, there was no difference when serum from splenectomized patients was employed in the presence or absence of exogenous tuftsin. Similar results were obtained under a variety of conditions, utilizing three different challenge particles with varying particle-cell ratios and serum from 20 different splenectomized patients. These results do not agree with the hypothesis that tuftsin plays a major role in promoting phagocytosis.     

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.

     

Effect of temperature on phagocytosis by human polymorphonuclear neutrophils.

Monolayers of human peripheral blood polymorphonuclear neutrophils and 14C-labeled Staphylococcus aureus were incubated at various temperatures. The rate and extent of phagocytosis was unchanged between 33 and 41 C but was depressed above and below this range     

Modulation of human polymorphonuclear leukocyte function by the flavonoid silybin

The effect in vitro of the naturally occurring flavonoid silybin on human polymorphonuclear leukocyte (PMN) functions has been studied. Preincubation of PMNs for 10 min at 37 degrees C with silybin inhibited, in a dose-dependent way, the luminol-enhanced chemiluminescence (CL) generated by stimulated cells without affecting the non-enhanced CL or superoxide anion production evaluated by the cytochrome C reduction assay. No significant effect of silybin on PMN phagocytic or chemotactic activities were found. Silybin did not absorb light at the wavelength of luminol-enhanced CL and was not toxic to PMNs at the concentrations used. Catalase, a scavenger of H2O2, inhibited luminol-enhanced CL to a similar degree as silybin; moreover, when incubated together with PMNs, silybin and catalase did not produce an additive inhibition of CL. On the contrary, the simultaneous addition of silybin and sodium azide, an inhibitor of myeloperoxidase, further increased inhibition over that seen with azide alone. These results suggest that inhibition of H2O2 may be the mechanism by which silybin inhibits the luminol-enhanced CL generated by stimulated PMNs. Such results indicate a possible anti-inflammatory activity for silybin even if their clinical relevance remains to be elucidated.     

Influence of encapsulation on staphylococcal opsonization and phagocytosis by human polymorphonuclear leukocytes

In previous studies, encapsulated Staphylococcus aureus strains have been shown to resist phagocytosis. In this investigation, the nature of the interference with phagocytosis by human polymorphonuclear leukocytes was examined by studying the opsonization of two pairs of unencapsulated (Smith compact and M variant) and encapsulated (Smith diffuse and M) S. aureus strains. The uptake of [3H]glycine-labeled bacteria by normal leukocytes was quantitatively measured after incubation of bacteria in pooled serum, C2-deficient serum, immunoglobulin-deficient serum, and serum from a rabbit immunized with S. aureus M. The presence of a capsule was found to interfere with opsonization by both the classical and alternative pathways of complement as well as by heat-stable opsonic factors in nonimmune human serum. This interference was significantly greater in the case of the S. aureus M strain than in the case of the Smith diffuse strain. The only effective opsonic source for S. aureus M was immune rabbit serum. It is proposed that encapsulation of S. aureus strains interferes with phagocytosis by preventing effective bacterial opsonization.     

Effect of rokitamycin on human polymorphonuclear leukocyte chemotaxis

The effect of rokitamycin on human polymorphonuclear leukocyte (PMNL) chemotaxis was studied in vitro and in vivo. It was found that rokitamycin in vitro at a concentration of 20 micrograms/ml caused a diminution of PMNL migration, while at lower concentrations no significant effects on migration was observed. In in-vivo studies before and after the ingestion of rokitamycin by six healthy individuals, no change on PMNL chemotaxis was found.     

Factors influencing the phagocytosis of Clostridium difficile by human polymorphonuclear leukocytes.

Phagocytosis of Clostridium difficile by human polymorphonuclear leukocytes (PMNs) and the possible role of the clostridial toxins in this process were investigated. Phagocytosis of C. difficile was independent of aerobiosis and clearly depended on opsonization. Either complement or antibodies to C. difficile could serve as opsonins. Toxigenic strains of C. difficile were more resistant to phagocytosis than were nontoxigenic strains. Pretreatment of PMNs with as much as 10,000 units of toxins from culture filtrates of C. difficile for 2 h had no effect on either the phagocytic activity of PMNs or their viability as determined by trypan blue exclusion. In contrast, treatment of human embryonic intestinal cells with the same amount of toxin under identical conditions resulted in cell death.     

Opsonization of yeast by human serum IgA anti-mannan antibodies and phagocytosis by human polymorphonuclear leucocytes.

The ability of sera from 72 patients with liver disease to opsonize yeast for phagocytosis by normal polymorphonuclear leucocytes has been studied. Seven showed defective opsonization. The opsonic activity of all but two sera was decreased markedly by heating at 56 degrees C for 1 h. When the two sera with heat stable opsonic activity were fractionated by gel filtration and by ion exchange chromatography, the activity copurified with IgA, not with IgG. The purified IgA, radiolabelled with 125I was shown to bind in a saturable manner to the yeast. Both sera had high levels of anti-yeast mannan IgA detected by an ELISA. In one case most of the anti-mannan activity was due to monomeric IgA, in the other it was dimeric. This was consistent with the observation of an apparent molecular weight of the opsonin of approximately 180 kD in one serum and 300-400 kD in the other.     

Heparin inhibits phagocytosis by polymorphonuclear leukocytes.

Phagocytosis of unopsonized Salmonella typhimurium 395, MR-10, opsonized Salmonella typhimurium 395 MS, and Staphylococcus epidermidis by rabbit polymorphonuclear leukocytes was inhibited by heparin at concentrations as low as 0.5 U/ml. Inhibition was dose dependent and nearly complete at 20 U/ml. Provided that heparin concentrations did not exceed 100 U/ml, inhibition could be largely reversed by washing. Heparin also reversibly inhibited the adherence of polymorphonuclear leukocytes to glass. In contrast, hexose monophosphate shunt activity of polymorphonuclear leukocytes stimulated by noningested S. typhimurium MR-10 or Streptococcus pyogenes B14 was not inhibited by heparin at concentrations as high as 100 U/ml.     

Inhibition of human polymorphonuclear leukocyte function by components of human colostrum and mature milk

To compare the effect of human colostrum (days 1 to 3 postpartum) and mature milk (days 170 ± 24 postpartum) on the function of polymorphonuclear leukocytes (PMNL), Ficoll-Hypaque-separated PMNL from the blood of 60 healthy volunteers were incubated with whole colostrum, colostral lipid, and colostral aqueous phase from 30 mothers, or with mature whole milk and its separated components from 30 mothers, and tested for resting and zymosan-stimulated oxidative metabolism, functional activity, and the presence of Fc receptors. Stimulated oxygen consumption, quantitative nitroblue tetrazolium dye reduction, [1-¹⁴C]glucose utilization, and Fc receptors were significantly (P < 0.05 to P < 0.001) less in PMNL exposed to whole human colostrum or colostral lipid than in non-lipid-exposed cells or cells exposed to the aqueous phase of colostrum. In contrast, PMNL exposed to whole mature milk or to its lipid or aqueous phase caused no significant decrease in any of these parameters when compared to nonexposed cells. In assays of phagocytosis, colostral PMNL or blood PMNL exposed to colostral lipid had a significant (P < 0.001) decrease in their ability to ingest [methyl-³H]thymidine-labeled Staphylococcus aureus when compared to non-lipid-exposed PMNL. Blood PMNL exposed to lipid from mature milk had no decrease in ability to ingest S. aureus. Analysis of total lipid and total and individual fatty acid content revealed a uniform increase in all components in mature milk when compared to colostrum. Lipid or lipid-soluble material present in human colostrum but not mature milk causes inhibition of phagocytosis and respiratory burst-related activities of PMNL.     

Electron microscopic study of phagocytosis of Escherichia coli by human polymorphonuclear leukocytes.

The fate of Escherichia coli strains within the polymorphonuclear leukocytes was studied by determining the killing of bacteria, measuring the release of degradation products, and examining the phagocytic bacteria by electron microscopy. When sufficiently opsonized, both unencapsulated and encapsulated E. coli strains were rapidly phagocytized by polymorphonuclear leukocytes. Once phagocytized, the two unencapsulated E. coli strains (K-12 and O111) were rapidly killed (99% of the bacteria were killed during the first 5 min of phagocytosis) and extensively degraded (about 40% of the radiolabeled material was released from bacteria after 15 min of phagocytosis). Electron micrographs taken after 15 min of phagocytosis revealed extensive structural changes in most of the internalized bacteria. In contrast to the rapid killing and extensive breakdown of these strains, encapsulated E. coli O78:K80 was more resistant to killing and withstood degradation by polymorphonuclear leukocytes (only 5% of the radioactivity was released from the radiolabeled bacteria after 1 h of phagocytosis). Electron micrographs of thin sections taken after 1 h of phagocytosis revealed virtually no structural changes. Most of the internalized bacteria were still surrounded by thick capsular material.     

Ammonium decreases human polymorphonuclear leukocyte cytoskeletal actin.

Ammonium, a weak base produced as a metabolic by-product of urea metabolism by bacterial pathogens, inhibits a variety of motile polymorphonuclear leukocyte (PMN) functions. It was initially assumed that the mechanism of leukocyte inhibition was due to cytoplasmic alkalinization. However, while it is clear that ammonium can effect cytoplasmic alkalinization, current data indicate that alterations in chemotaxis, degranulation, and receptor recycling occur independently of cytoplasmic alkalinization. Since these are motility-related events, we examined the possibility that alterations in cytoskeletal actin may account for the effects of ammonium on PMN function. The results indicate that ammonium can inhibit degranulation, decrease cytoskeletal actin, and increase actin depolymerization rates. These findings are supported by five lines of evidence. First, formylmethionyl-leucyl-phenylalanine (fMLP)-induced elastase release was inhibited by 85% +/- 3% in the presence of ammonium, and ammonium by itself did not stimulate elastase release. Second, ammonium treatment of resting PMNs caused a rapid 38% +/- 6% decrease in cytoskeletal actin. Third, ammonium treatment accelerated the fMLP-induced depolymerization phase of the cytoskeletal actin transient by 150% +/- 12%. Fourth, in resting PMNs treated with cytochalasin B or D, ammonium induced a 21% +/- 4% and a 25% +/- 5% decrease in cytoskeletal actin, respectively. Conversely, ammonium did not affect the ability of the cytochalasins to inhibit an fMLP-induced cytoskeletal actin transient. Fifth, pertussis toxin treatment of neutrophils did not affect the ammonium-stimulated decrease in cytoskeletal actin. These results suggest that ammonium can inhibit neutrophil function by altering cytoskeletal actin and therefore provide new information regarding potential pathogenic mechanisms for bacterial pathogens.     

Quantitative phagocytosis by human polymorphonuclear leucocytes. Use of radiolabelled emulsions to measure thae rate of phagocytosis.

A new micro-method for the quantitative measurement of phagocytosis by neutrophils is described. The material used for phagocytosis consists of a radioactive oil emulsion coated with E. coli lipopolysaccharide. Uptake of radioactive material is a function of cell number, duration of incubation, dilution of serum used for opsonization, content of lipopolysaccharide and concentration of emulsion. This method can be used to quantify rapidly and precisely phagocytosis rates of as few as 5 x 10(4)-10(6) polymorphonuclear leucocytes and the opsonic activity of 10 microliter serum.     

Effect of staphylococcal alpha-toxin on phagocytosis of staphylococci by human polymorphonuclear leukocytes.

Forty species of anaerobes were screened for the ability to produce an ether-extractable mutagen which is present in the feces of 15 to 20% of individuals in populations at high risk for colon cancer. This mutagen can be produced in vitro by incubating the feces of these individuals anaerobically or by supplementing anaerobic broths with methanol extracts of the feces and incubating them with a dilute fecal inoculum. Of the anaerobes screened, strains of five species of Bacteroides (B. thetaiotaomicron, B. fragilis, B. ovatus, B. uniformis, and Bacteroides group 3452A) were capable of producing five- to eightfold increases in the concentration of mutagen. For in vitro production in broth, all producers required bile and the methanol extract for feces from a person who excretes the mutagen. Mutagen production appeared to be constitutive and occurred during the stationary phase of growth. Cell-free extracts were active and produced mutagen considerably faster than did whole cells. Our observations indicate that the excretion of this mutagen by certain people is dependent on the presence of some precursor of unknown origin. The mutagen-producing species of bacteria are among the most common of the intestinal microflora and were present in mutagen excreters and nonexcreters as well.     

Trypanosoma cruzi: sequence of phagocytosis and cytotoxicity by human polymorphonuclear leucocytes.

We have studied the relationship between phagocytosis and cytotoxicity of human polymorphonuclear leucocytes (PMN) to sensitized Trypanosoma cruzi. Assays were done simultaneously using [3H]-uridine labelled epimastigotes as target cells. Phagocytosis was evaluated by the uptake and cytotoxicity by the release of parasite associated [3H]-uridine. Both reactions reached maximum levels at the same effector- to target-cell ratio and antibody concentration. Uptake of epimastigotes by PMN was highest at 30 min and intracellular disruption and release of parasite debris took place later. In conditions that precluded repeated uptake of sensitized radiolabelled T. cruzi, the release profile of [3H]-uridine from PMN that contained intracellular parasites was similar to that of the standard cytotoxic assay. However, as the ingestion phase was separated from the release step, no lag in the onset of the reaction was observed. Although we cannot rule out extracellular killing, the results of this study demonstrate that the bulk of damaged T. cruzi epimastigotes had been previously internalized by the PMN.     

Aggregation of human polymorphonuclear leucocytes during phagocytosis of bacteria.

The process of aggregation of human polymorphonuclear leucocytes (PMN) during the uptake of bacteria was studied. Radiolabelled S. aureus were opsonized in different sera, washed, resuspended in buffer and added to the PMN. Uptake of the bacteria and aggregation of the PMN were measured simultaneously. Maximal aggregation occurred within 6 min, when 5 X 10(6) PMN had phagocytosed 2.5 X 10(8) S. aureus. Also the effects of serum concentrations and different sera for opsonization of the bacteria on PMN aggregation were studied. Despite normal uptake, aggregation of PMN was low when bacteria were opsonized in complement-deficient sera. Furthermore when PMN were treated with pronase to inactivate complement receptors on the cell surface of the PMN, and bacteria preopsonized in immune serum were added, no change in uptake occurred, although the degree of aggregation halved compared to control PMN. So, interaction between the bacteria and the complement receptor of the PMN cell membrane is needed for triggering the process of aggregation. By using dansylcadaverin and diphenylamine to modulate lysosomal enzyme release, azide or PMN from a chronic granulomatous disease patient to study the effect of the formation of oxygen species, and theophylline, DB-cAMP or 8 Br-cAMP to increase cAMP levels, it was concluded that aggregation of PMN during phagocytosis was not dependent on oxygen metabolism, degranulation or cAMP levels of PMN.     

The polymorphonuclear leukocyte

Polymorphonuclear leukocytes (PMNs) are one of the main sources of enzymes responsible for tissue damage in inflammatory processes. These enzymes are stored in two types of cytoplasmic granules. Azurophil granules contain lysosomal hydrolases, neutral serine proteinases, and bactericidal elements (myeloperoxidase and lysozyme). Specific granules contain collagenase, lysozyme and lactoferrin but lack lysosomal hydrolases. PMNs store all four classes of tissue proteinases, carboxyl, thiol and serine proteinases in the azurophil granules, and metallo proteinases in the specific granules. Three serine proteinases have been identified, elastase, cathepsin G and a third enzyme, which together account for a large proportion of the protein of the azurophil granules. In the course of phagocytic events, all these enzymes are released extracellularly. The neutral proteinases degrade proteoglycans and collagen. In vitro, they stimulate B-lymphocytes, which suggests that they may have immuno-potentiating activity when they are released at sites of chronic inflammation.