Isolation and Partial Characterization of a Staphylococcal Leukocyte Cytotaxin

A factor which attracts rabbit polymorphonuclear leukocytes both in vitro and in vivo was isolated from the culture filtrate of a strain of Staphylococcus aureus. The activity of the chemotactic factor was independent of fresh serum and it was nondialyzable. Incubation of the factor with heat-inactivated human serum markedly inhibited its chemotactic property. The factor was heat-labile (80 C, 10 min) in the crude culture filtrate but was heat-stable when partially purified.     

Bilirubin inhibits the chemotactic activity of human polymorphonuclear leukocytesin vitro

Using the migration of human polymorphonuclear leukocytes in agarosein vitro, it was established that bilirubin inhibits migration (chemotaxis) of these cells upon stimulation with both complement-derived (zymosan-activated serum) and bacteria-derived (abacterial filtrate ofE. coli) chemotactic agents.     

Changes in the migratory activity of polymorphonuclear leukocytes after administration of Freund’s adjuvant

Spontaneous and chemotactic activity of polymorphonuclear leukocytes, obtained from the peritoneal cavity, was studied after administration of complete and incomplete Freund's adjuvant to rabbits at intervals of 18-26 d after adjuvant injection. Whereas injection of both complete and incomplete adjuvant increased spontaneous migration of rabbit polymorphonuclear leukocytes, the migration directed by the chemotactic signal, induced in fresh serum by E. coli endotoxin, was decreased. The chemotaxigenic activity of two factors, isolated from cell walls of Listeria monocytogenes was also tested: factor Ei (its most potent component is lipopolysaccharide) and a purified phenol extract of lipopeptidopolysaccharide nature.     

Binding of protein chemotactic factors to the surfaces of neutrophil leukocytes and its modification with lipid-specific bacterial toxins

Summary The binding to neutrophil leukocytes of human serum albumin (HSA), which is chemokinetic for leukocytes, i.e. influences their rate of locomotion, and of alkali-denatured HSA, which is chemotactic for leukocytes, i.e. influences their direction of locomotion, was studied. Native serum albumin showed low affinity binding to the neutrophil surface. Denatured serum albumin showed saturable binding with a K_a of approximately 10⁶ litres per mole to about 10⁶ binding sites per cell. Another protein chemotactic factor, _s-casein, gave similar binding. These results exclude that chemotactic reactions to denatured proteins are mediated in a completely non-specific manner and suggest the presence on the cell of a restricted number of defined recognition sites. Binding was reduced following treatment of the cells with either of two lipid-specific bacterial toxins, perfringolysin, the -toxin of Clostridium perfringens, an oxygen-labile cholesterol-specific toxin, and Staphylococcus aureus Sphingomyelinase C. Both have previously been shown to reduce chemotactic reactions and both were used at doses which did not reduce cell viability. These results suggest an important, and possibly direct, role for membrane lipid in the binding sites for chemotactic factors. Visual analysis of the behaviour of perfringolysin-treated neutrophils showed that these cells were still capable of chemotactic locomotion. The cells appeared to be less efficient than normal in detecting chemotactic gradients only when at a distance from the gradient source, a finding which is consistent with reduced binding of the chemotactic factor to the cell surface.     

Effect of blood serum, caffeine and heparin on in vitro phagocytosis of frozen-thawed bull sperm by neutrophils derived from the peripheral blood of cows

Although polymorphonuclear leukocytes (PMNs) are recruited into the uterine lumen to phagocytize sperm, factors controlling the phagocytotic ability of PMNs in cattle are not well documented. The objective was to determine the effects of blood serum, caffeine, and heparin on chemotaxis of PMNs for sperm and phagocytosis of sperm by PMNs in cows. Polymorphonuclear leukocytes were obtained (centrifugation) from a cow's peripheral blood. In Experiment 1, the chemotactic activity of PMNs increased (P < 0.01) when fresh serum was included in the medium (1226 cells/mm² in serum vs. 1110 cells/mm² in BSA), regardless of the presence of sperm, whereas heat-inactivated serum (1099 cells/mm²) did not affect their activity (P = 0.65). Phagocytosis of live and dead sperm by PMNs both increased (P < 0.01) in the presence of fresh serum (incidences of 54.5 and 48.0%, respectively), but stimulation was decreased (P < 0.01) by supplementation of the medium with ≥1 mM caffeine (20.6-30.3%). Serum-stimulated chemotactic activity of PMNs (1218 cells/mm²) was also decreased (P < 0.01) in the presence of caffeine (1090 cells/mm²). Furthermore, supplementation of PMNs with heparin in the presence of serum decreased (P < 0.01) both phagocytotic (from 43.8% to 21.5-31.7%) and chemotactic activities of PMNs (from 1124 to 1048-1108 cells/mm²). We inferred that opsonization in the presence of active complement stimulated phagocytotic and chemotactic activities of PMNs, and that both caffeine and heparin decreased serum-stimulated phagocytotic and chemotactic activities of PMNs.     

Cytolytic Plasma Factor in Experimental Coccidioidomycosis

Peripheral blood leukocytes from guinea pigs infected with Coccidioides immitis 5 weeks previously were lysed significantly when incubated at 37 C for 3.5 hr with a concentrated C. immitis culture filtrate, but not with a concentrated Aspergillus fumigatus or Blastomyces dermatitidis culture filtrate. Mononuclear leukocytes and presumably granulocytes were both lysed. Characterization of the factor responsible for this leukocytolysis reaction revealed that it was contained in plasma and serum but not in leukocytes from infected guinea pigs. It was complement-dependent, active after heating at 56 C, and stable after storage at -20 C for as long as 14 months.     

A quantitative and qualitative study of blood monocytes in patients with bronchogenic carcinoma

Summary Absolute circulating number and functions of blood monocytes (i.e., pinocytosis, phagocytosis, and chemotaxis) were studied in 25 patients with untreated bronchogenic carcinoma and in 28 control subjects. The absolute circulating monocyte count was increased in 20 (80%) of the patients. There was no difference in the pinocytic and phagocytic activity of patient and control monocytes. In contrast, patient monocytes showed depressed chemotactic responsiveness. This defect was more severe in small cell anaplastic carcinoma than in the other histologic types of bronchogenic carcinoma (P=0.001), and may explain the difference in macrophage infiltration seen in solid tumours of the lung. There was no correlation between chemotaxis and clinical stage. Depressed chemotaxis may be related to a plasma factor, since patient plasma inhibited the chemotaxis of control monocytes as well as the activity of chemotactic agents. The defective chemotaxis and the presence of plasma inhibitory activity may interfere with the ability of blood monocytes to accumulate as macrophages in tumour sites. Abbreviations used in this paper are: MCR, monocyte chemotactic response; SAC, small cell anaplastic bronchogenic carcinoma; OBC, non-small cell bronchogenic carcinoma MEM, Eagle's minimal essential medium; CFI, chemotactic factor inhibitor(s); HSA, human serum albumin     

Skin-window study on the migration of leukocytes of newborns and infants

Migration of leukocytes of newborns and of infants up to the age of 6 months was studied using the in vivo skin-window technique according to Rebuck. Using the non-specific stimulation (abrasion of the skin only) a slight age-dependent physiological increase of migration of cells was observed within the observation period; after a strong local irritation with diphtheria-tetanus-pertussis vaccine (Alditepera) there was a vigorous migratory response of cells in the skin lesion. Both quantitatively and qualitatively, the migration pattern (i.e. shift from PMN leukocyte to mononuclear cells in the exudate within a 1 d period after abrasion) was not influenced by immunization of infants with Alditepera, suggesting thus the nonspecific nature of this cellular response. The "normal" values of the chemotactic response of leukocytes of newborns and infants are given as a basis for evaluation of defects of this functional activity of leukocytes.     

IL-8 production by human polymorphonuclear leukocytes. The chemoattractant formyl-methionyl-leucyl-phenylalanine induces the gene expression and release of IL-8 through a pertussis toxin-sensitive pathway.

IL-8 is a novel chemotactic cytokine, produced by a variety of blood and tissue cells, that has marked activating effects on polymorphonuclear leukocytes (PMN). We report that IL-8 is produced and released by human PMN after stimulation with the chemotactic agonist FMLP. Release of IL-8 in response to FMLP was transient and not influenced by PMN adherence or by the absence of serum in the medium. Maximum yields were usually obtained with 10 nM FMLP within 2 h of stimulation (0.5-3.5 ng/ml/7 x 10(6) cells, range of 17 different donors). IL-8 release was dependent on FMLP-induced de novo protein synthesis because it was inhibited by cycloheximide, was paralleled by enhanced expression of IL-8 mRNA and was potentiated from two- to sixfold after preincubation of PMN with cytochalasin B. The FMLP effect was direct and not dependent on LPS or on contaminating monocytes, which showed only low responsiveness to FMLP. Pretreatment of PMN with pertussis toxin prevented FMLP-dependent IL-8 production, the effect being evident both at the level of mRNA expression and protein secretion. In addition, two other chemoattractans, platelet-activating factor and C5a, were found capable to induce release of IL-8 by PMN. The results of this study suggest that chemotactically stimulated PMN may be able to amplify the recruitment process of PMN to the inflammatory site by releasing IL-8. As a long-lived cytokine, IL-8 could markedly prolong the attractant effect.     

The antiinflammatory effects of essential fatty acid deficiency in experimental glomerulonephritis. The modulation of macrophage migration and eicosanoid metabolism

Dietary polyunsaturated fatty acid modulation exerts a beneficial effect in immune-mediated glomerulonephritis. To elucidate the mechanisms underlying this phenomenon, the effects of essential fatty acid (EFA) deficiency on the heterologous phase of nephrotoxic nephritis in rats (induced by the injection of a rabbit antiglomerular basement membrane antibody) were studied. The heterologous phase of nephrotoxic nephritis was characterized by an invasion of leukocytes into the glomerulus. Polymorphonuclear neutrophils predominated early on (3 h), whereas macrophages predominated at 24 and 72 h. EFA deficiency selectively prevented the influx of macrophages into the glomerulus. The invasion of polymorphonuclear neutrophils, in contrast, was unaffected. The influx of leukocytes into the glomerulus during nephritis was accompanied by a marked enhancement (10- to 40-fold) in glomerular thromboxane and leukotriene B4 production. EFA deficiency largely attenuated this change. Renal dysfunction during the heterologous phase of nephritis was manifested as azotemia, polyuria, sodium retention, and proteinuria. With EFA deficiency, polyuria, azotemia, and sodium retention were not seen. Proteinuria was reduced by approximately 85%. To address whether the lack of macrophage migration into the glomerulus in the context of nephritis with EFA deficiency might be due to a functional defect in macrophage migration, the chemotactic responsiveness of EFA-deficient macrophages was examined. EFA-deficient macrophages displayed normal chemotactic migration toward activated C. In sum, EFA deficiency prevents the invasion of macrophages into the glomerulus in nephrotoxic nephritis and attenuates the accompanying metabolic and functional alterations, but does not affect macrophage chemotactic responsiveness. Alterations in macrophage elicitation and lipid mediator generation by inflamed glomeruli thus appear to be central to the salutary effect of dietary polyunsaturated fatty acid modification on glomerulonephritis.     

Monocyte chemotaxis in patients with nonseminomatous testicular carcinoma

Summary The chemotactic responsiveness of blood monocytes was tested in 16 patients with nonseminomatous testicular carcinoma before, during, and after chemotherapy. All the patients initially had monocyte chemotaxis within the normal range. No correlation with the histology of the tumor, the clinical stage, or the presence in serum of -fetoprotein and human chorionic gonadotropin was observed. Plasma from the patients did not inhibit the chemotaxis of normal monocytes, and serum from the patients contained no chemotactic factor inhibitor. During intensive chemotherapy with cis-platinum, bleomycin, and vinblastine a reversible defect in chemotaxis occurred without correlation to the development of fever. Two months after the completion of chemotherapy the chemotactic responsiveness was unchanged compared with pretreatment values. In conclusion, this study shows normal monocyte chemotaxis in patients with testicular carcinoma, which is in contrast to reports on a variety of other solid tumors.     

Factor XIII-dependent generation of 5th complement component(C5)-derived monocyte chemotactic factor coinciding with plasma clotting.

Blood coagulation or plasma clotting caused generation of a monocyte chemotactic factor(s) in vitro. The chemotactic factor, of which the apparent molecular mass was 75 kDa, shared antigenicity with complement C5 and possessed the affinity to monocytes, but not to polymorphonuclear leukocytes. The generation of the chemotactic factor was hindered in the presence of a thiol enzyme inhibitor, p-chloromercuriphenyl sulfonic acid, at the concentration of 1 mmol/l, although the gelation of plasma was apparently completed. Furthermore, the generation of chemotactic factor was not observed when a plasma deficient in blood coagulation factor XIII, which is a precursor of a thiol enzyme, plasma transglutaminase, was used; and the activity normally appeared when the deficient plasma was reconstituted with purified factor XIII or with a tissue transglutaminase prior to clotting. When the human sera were injected into guinea pig skin, the serum derived from normal plasma or from the reconstituted factor XIII deficient one caused mononuclear cell infiltration, however, the serum from the deficient plasma without reconstitution infiltrated to a significantly smaller extent. These results indicated that the complement system was initiated somehow during the clotting process resulting in the generation of the C5-derived monocyte chemotactic factor in cooperation with factor XIIIa (activated factor XIII).     

Chemotaxis of neutrophil leukocytes towards substratum-bound protein attractants

Human blood neutrophil leukocytes were shown by the checkerboard filter assay to be capable of chemotactic migration in response to gradients of filter-bound chemotactic proteins in the absence of protein in free solution. Chemotactic proteins (casein, denatured serum albumin) and chemokinetic proteins (native serum albumin) bind substantially to substrata such as filters and glass, whereas low molecular weight chemotactic factors such as formyl-methionyl-phenylalanine bind poorly. Cells do not locomote towards the latter factors in the absence of protein but can, when migrating on substratum-bound albumin, respond chemotactically to gradients of fluid-phase formyl peptide.     

Group B streptococci inhibit the chemotactic activity of the fifth component of complement

Infection with group B streptococci (GBS) is associated with a poor acute inflammatory response in which neutrophils fail to localize at the site of invasion. In the present studies, we have examined the effects of group B streptococci on C-derived chemotactic activity in human serum. Fresh human serum was activated to form C5a and C5adesarg by incubation with zymosan. The activated serum was then incubated with group B organisms, centrifuged, and the supernatants tested for chemotactic activity for human polymorphonuclear leukocytes. Group B organisms caused a dose-dependent decrease in C-dependent chemotactic activity. The degree of inhibition was profound with 1 X 10(9) bacteria/ml (10% of control). Experiments indicated that significant chemotactic factor inactivation occurred within 2 min of exposure to GBS organisms, while maximal inhibition occurred after 30 min incubation. A number of different strains of GBS of types I, II, and III possessed inhibitory activity. In contrast, group D streptococci, Staphylococcus aureus, Escherichia coli and Klebsiella pneumoniae failed to inhibit the C-derived chemotactic activity in human serum. Group A streptococci that were M protein positive also inactivated C-dependent chemotactic activity in serum, as previously reported. The inhibitory activity of the GBS strains could be abolished by heat or trypsin treatment but not by neuraminidase, pronase, or pepsin. C5a levels in zymosan-activated serum as measured by RIA were not decreased after incubation with an inhibitory strain suggesting that absorption was not involved. SDS-PAGE analysis revealed that group B streptococci degrade the C5a molecule, increasing its electrophoretic mobility by removing a fragment with a m.w. of approximately 650 Da. Thus, one of the reasons for the poor inflammatory response at the site of GBS infection may reside in the ability of these pathogens to inactivate C-derived inflammatory mediators. The GBS C5a-ase activity probably serves as an additional virulence factor for these organisms contributing to the poor inflammatory response characteristic of group B streptococcal infection.     

Cell polarity: an examination of its behavioral expression and its consequences for polymorphonuclear leukocyte chemotaxis

Locomoting polymorphonuclear leukocytes (PMNs) exhibit a morphological polarity. We demonstrate that they also exhibit a behavioral polarity in their responsiveness to chemotactic factor stimulation. This is demonstrated by (a) the pattern of their locomotion in a homogeneous concentration of chemotactic factors, (b) their responses to increases in the homogeneous concentration of chemotactic factors, and (c) their responses to changes in the direction of a chemotactic gradient. The behavioral polarity is not a function of the rate of locomotion of the particular stimulant used to orient the cells, but may reflect an asymmetric distribution of chemotactic receptors or the motile machinery. The polar behavior affects the chemotactic ability of PMNs. The data are discussed in relation to possible mechanisms of sensing a chemotactic gradient.     

Inhibition of leukocyte function by serum from patients with trichinellosis

Modification of leukocytic function has been reported in only a few human parasitic diseases. In this study we evaluated the effects of the sera from patients infected with Trichinella on chemotactic and phagocytic responses in leukocytes. Leukocyte chemotaxis was tested by the agarose method and phagocytosis by the technique of Yamamura, modified for Saccharomyces cerevisiae. Sera were acquired from patients during a trichinellosis outbreak that occurred in northern Italy in 1986. The parasite was isolated from 1 patient and isoenzymatically typed as Trichinella sp. 3, a new taxon, previously considered Trichinella nelsoni. The results indicated that sera from Trichinella-infected humans inhibited both chemotaxis and phagocytic responses in leukocytes. These findings suggest the existence of serum factor(s) in trichinellosis patients that modify host leukocytic functions. The source and nature of active serum components and the mechanism by which they modulate leukocyte function remain to be clarified.     

Temporal adaptation of neutrophil oxidative responsiveness to n-formyl-methionyl-leucyl-phenylalanine. Acceleration by granulocyte-macrophage colony stimulating factor

This investigation was undertaken to clarify the mechanism by which purified recombinant human granulocyte-macrophage colony stimulating factor (GM-CSF) potentiates neutrophil oxidative responses triggered by the chemotactic peptide, FMLP. Previous studies have shown that GM-CSF priming of neutrophil responses to FMLP is induced relatively slowly, requiring 90 to 120 min of incubation in vitro, is not associated with increased levels of cytoplasmic free Ca2+, but is associated with up-regulation of cell-surface FMLP receptors. We have confirmed these findings and further characterized the process of GM-CSF priming. We found that the effect of GM-CSF on neutrophil oxidative responsiveness was induced in a temperature-dependent manner and was not reversed when the cells were washed extensively to remove the growth factor before stimulation with FMLP. Extracellular Ca2+ was not required for functional enhancement by GM-CSF and GM-CSF alone effected no detectable alteration in the 32P-labeled phospholipid content of neutrophils during incubation in vitro. Our data indicate that GM-CSF exerts its influence on neutrophils by accelerating a process that occurs spontaneously and results in up-regulation of both cell-surface FMLP receptors and oxidative responsiveness to FMLP. Thus, the results demonstrate that, with respect to oxidative activation, circulating endstage polymorphonuclear leukocytes are nonresponsive or hyporesponsive to FMLP; functional responsiveness increases dramatically as surface FMLP receptors are gradually deployed after the cells leave the circulation. Thus, as neutrophils mature, their responsiveness to FMLP changes in a manner which may be crucial for efficient host defense. At 37 degrees C, this process is markedly potentiated by GM-CSF. We conclude that endogenous GM-CSF, released systemically or at sites of infection and inflammation, potentially plays an important role in host defense by accelerating functional maturation of responding polymorphonuclear leukocytes.     

Molecular mechanism of desensitization of the chemokine receptor CCR-5: receptor signaling and internalization are dissociable from its role as an HIV-1 co-receptor.

The chemokine receptor, CCR-5, a G protein-coupled receptor (GPCR) which mediates chemotactic responses of certain leukocytes, has been shown to serve as the primary co-receptor for macrophage-tropic human immunodeficiency virus type 1 (HIV-1). Here we describe functional coupling of CCR-5 to inhibition of forskolin-stimulated cAMP formation via a pertussis toxin-sensitive G(i) protein mechanism in transfected HEK 293 cells. In response to chemokines, CCR-5 was desensitized, phosphorylated and sequestered like a prototypic GPCR only following overexpression of G protein-coupled receptor kinases (GRKs) and beta-arrestins in HEK 293 cells. The lack of CCR-5 desensitization in HEK 293 cells in the absence of GRK overexpression suggests that differences in cellular complements of GRK and/or beta-arrestin proteins could represent an important mechanism determining cellular responsiveness. When tested, the activity of CCR-5 as an HIV-1 co-receptor was dependent neither upon its ability to signal nor its ability to be desensitized and internalized following agonist stimulation. Thus, while chemokine-promoted cellular signaling, phosphorylation and internalization of CCR-5 may play an important role in regulation of chemotactic responses in leukocytes, these functions are dissociable from its HIV-1 co-receptor function.     

Tumor necrosis factor is chemotactic for monocytes and polymorphonuclear leukocytes

Human recombinant tumor necrosis factor (TNF) induced migration across polycarbonate and nitrocellulose filters of human peripheral blood monocytes and polymorphonuclear leukocytes, TNF was active in inducing migration at concentrations less than 1 U/ml, and maximal responses (observed at greater than 100 U/ml) were comparable to those elicited by standard reference chemoattractants (FMLP, 10 nM; activated human serum, 5%). Checkerboard analysis performed by seeding different concentrations of TNF above and below the filter revealed that maximal induction of migration required a positive concentration gradient between the lower and upper compartments and that TNF elicited an actual chemotactic response in phagocytes. An anti-TNF rabbit antiserum and anti-TNF mouse monoclonal antibody abolished the chemotactic activity of TNF. Recombinant lymphotoxin was also chemotactic for phagocytes, and its activity was blocked by an anti-lymphotoxin antiserum. Human umbilical vein endothelial cells and blood large granular lymphocytes did not respond chemotactically to TNF under conditions in which appropriate reference chemoattractants were active. The chemotactic activity of TNF may serve to recruit phagocytic cells from the blood compartment to amplify resistance against noxious agents.     

Asymmetric distribution of the chemotactic peptide receptor on polymorphonuclear leukocytes

The distribution of chemotactic peptide receptors on polymorphonuclear leukocytes (PMNs) was visualized using tritiated chemotactic peptide, N- formylmethionyl-leucylphenylalanine, coupled to hemocyanin (HY-FMLP). This probe was biologically active and the number of HY-FMLP molecules bound to the cell in a saturable manner corresponded closely to the number of peptide receptors characterized for rabbit peritoneal polymorphonuclear leukocytes (Sullivan, S. J., and S. H. Zigmond, 1980, J. Cell Biol., 85:703-711). Cells exhibiting locomotion have a polar morphology easily recognized in the scanning electron microscope. HY- FMLP bound to these cells was asymmetrically distributed with the highest density of HY-FMLP bound to the midregion of the cell. There were very few particles bound to the tail regions. The binding to the leading ruffles was variable but usually less than to the midregion. Addition of high concentrations of uncoupled FMLP eliminated HY-FMLP binding, confirming that the hemocyanin observed was a marker for the saturable chemotactic peptide receptor. The asymmetry in receptor distribution was seen on cells that had been stimulated by low concentrations of either FMLP or another chemotactic factor, leukotriene B4. Thus, peptide binding to the receptor was not required for the development of the asymmetric distribution. The low density of receptors in the tail region of the cell was consistent with the decreased responsiveness of the tail to chemotactic stimulation (Zigmond, S. H., H. I. Levitsky, and B. J. Kreel, 1981, J. Cell Biol., 89:585-592). The receptor asymmetry may contribute to the polar behavior exhibited by polymorphonuclear leukocytes and would be expected to quantitatively modify the directional information available to a cell in a gradient of chemotactic peptide.