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Chemotaxis of human polymorphonuclears in vitro. V. Role of the nonsegmented neutrophils and of the experimental conditions in the impairment of chemotaxis observed during bacterial infections.

Chemotaxis of human leukocytes was studied in vitro with a microfilter having pores of 3 micrometer used as a substrate for the gradient. Under these conditions, nonsegmented neutrophils did not reach the compartment filled with the attractant, but a significant proportion of them did so when filters with larger pores were substituted. When leukocytes from infected patients were tested with the usual 3 micrometer pore filters, chemotaxis was reduced (as previously shown), but less markedly and less frequently in simultaneous experiments with larger pores. In experiments performed under agarose layers instead of filters, nonsegmented neutrophils responded normally to chemoattraction, again suggesting that their impaired migration in filter experiments was a matter of pore size. When leukocytes from infected patients were assayed under agarose, no impairment occurred; on the contrary, a slight increase in both chemotaxis and random motility was observed. It was therefore concluded that some published cases of impaired neutrophil chemotaxis in infection might be due to technical bias related to pore size.

Bacterial Infections

Chemotaxis under agarose: a new and simple method for measuring chemotaxis and spontaneous migration of human polymorphonuclear leukocytes and monocytes.

A variety of methods have been devised for the study of spontaneous and directed cell migration. Among these, the membrane filter method introduced by Boyden in 1962, with its more recent modifications, has become the technique of choice for studies of leukocyte migration in vitro. This method, however, cannot be applied without alteration to studies of chemotaxis and spontaneous migration of cells of different types. We describe in this report a new and simple method for studying human leukocyte chemotaxis, in vitro, which is based upon migration of cells under agarose gel. This method has application to both polymorphonuclear leukocytes and monocytes, permits measurement of both chemotaxis and spontaneous migration, requires fewer cells per test, and is rapid, simple, reproducible, and inexpensive to set up.

Cell Movement

Casein-mediated neutrophil chemotaxis: a parallel between surface binding and chemotaxis.

This study was directed toward demonstrating an interaction between human polymorphonuclear leukocytes (PMNs) and the chemotactic factor,casein. Experiments conducted using fluorescein-labeled casein indicated that PMNs have a membrane receptor for this chemotactic factor. This receptor was specific for casein and was not found on lymphocytes. A direct parallel was shown between the binding of FITC--casein and the PMN chemotactic response to this protein. Binding to the receptor was optimal at 25 degrees C, unaffected by sodium azide, and partially reduced by iodoacetate. Enzymatic treatment of PMNs with pronase and trypsin resulted in a loss of both FITC--casein-mediated PMN fluorescence and chemotaxis. Our data indicate that human PMNs have a membrane receptor for casein and that interaction with this receptor results in a chemotactic response.

Binding, Competitive

Studies on bacterial chemotaxis. II. Effect of cheB and cheZ mutations on the methylation of methyl-accepting chemotaxis protein of Escherichia coli.

Radioactive proteins from chemotactic mutants of Escherichia coli with continuous tumbling phenotype (cheB and cheZ) and their otherwise isogenic parent were compared by two-dimensional gel electrophoresis. The system was capable of separating non-methylated methyl-accepting chemotaxis protein (MCP) from its methylated equivalent. The analysis of proteins from the envelope fraction of the bacteria showed that the cheB mutants contained a larger portion of methylated MCP than did the parent. However, the change of MCP methylation level was small, if any, in cheZ strains. The results suggest that the product of cheB gene and the product of cheZ gene are not functional complementary. The product of cheB gene functions in controlling the level of methylation at the stationary state of the organisms. In addition to known MCP species, a new MCP of about 43,000 daltons was found. This MCP appears to be involved in transducing signals of some sugars.

Bacterial Proteins

Studies of bacterial chemotaxis in defined concentration gradients. A model for chemotaxis toward L-serine.

The details of the chemotactic response of Salmonella typhimurium to gradients of L-serine have been examined in some detail. Two relatively macroscopic techniques have been employed to measure the bacterial response. These include measurements of the average velocity as the bacterial population moves toward attractants, and measurement of the upward-to-downward flux ratio, R, in the stable preformed attractant gradients. The dependence of the average velocity on gradient appears to be hyperbolic in nature, while the flux ratio depends linearly on the gradient. These data suggest a microscopic model for the dependence of bacterial behavior on the serine gradient. The model involves a linear dependence of the mean lifetime of a bacterial trajectory on the gradient for those bacteria moving toward higher attractant concentration. Those moving toward low concentrations of attractant do not change the mean duration of their trajectories, or the speed at which a given bacterium swims through the solution. This model generates the observed dependences of the average velocity and flux ratio on gradient. Interpretation of the experimental data suggests that a gradient which increases serine concentration by a factor of 2 in 10 mm is sufficient to double the average duration of a trajectory for a bacterium moving directly up the gradient. The concentration dependence of the chemotactic response to serine is more complicated. It suggests that more than one receptor of serine may be involved in determining chemotactic behavior to this attractant.

Chemotaxis

Neutrophil function in anergic surgical patients: neutrophil adherence and chemotaxis.

Skin test anergy (A) to recall antigens identifies surgical patients at high risk for sepsis. We studied neutrophil function in such patients to assess any alteration in their host defense mechanisms. Neutrophil adherence was measured with a modified adherence assay capable of measuring the adherence of neutrophils in whole blood or purified neutrophil suspensions, and neutrophil chemotaxis was assessed by the Boyeden technique. Twenty-one laboratory controls had a neutrophil adherence of 71.5 +/- 3.8% (mean +/-SD) and chemotaxis of 128.1 +/- 2.4,micro (mean +/-SD). Fifty-four hospitalized patients with normal skin tests had neutrophil adherence of 72.5 +/- 13.1% (p ~ 0.5 relative to control) and chemotaxis of 123.3 +/- 3.1 micro (p ~ 0.5). Twenty three relatively anergic patients had values of 84.3 +/- 7.9% (p < 0.001) and 103.7 +/- 2.0 micro (p < 0.001). Forty five A patients had adherence of 85.0 +/- 7.0% (p < 0.001) and chemotaxis of 90.4 +/- 2.9 micro (p < 0.001). The correlation coefficient between increased neutrophil adherence and decreased chemotaxis r = 0.81 has p < 0.0005. A factor which increased the adherence of normal control neutrophils was found in the plasma but not the serum of anergic patients. Inhibitors of control neutrophil chemotaxis have been shown in both serum and plasma of patients with decreased autologous neutrophil chemotaxis. We propose that this altered neutrophil function (possibly with other defects) in anergic patients may compromise their host defenses and render them susceptible to infection.

Adolescent

Chemotaxis by Pseudomonas aeruginosa.

Chemotaxis by Pseudomonas aeruginosa RM46 has been studied, and conditions required for chemotaxis have been defined, by using the Adler capillary assay technique. Several amino acids, organic acids, and glucose were shown to be attractants of varying effectiveness for this organism. Ethylenediaminetetraacetic acid was absolutely required for chemotaxis, and magnesium was also necessary for a maximum response. Serine taxis was greatest when the chemotaxis medium contained 1.5 X 10(-5) M ethylenediaminetetraacetic acid and 0.005 M magnesium chloride. It was not necessary to include methionine in the chemotaxis medium. The strength of the chemotactic responses to glucose and to citrate was dependent on prior growth of the bacteria on glucose and citrate, respectively. Accumulation in response to serine was inhibited by the addition of succinate, citrate, malate, glucose, pyruvate, or methionine to the chemotaxis medium. Inhibition by succinate was not dependent on the concentration of attractant in the capillary. However, the degree to which glucose and citrate inhibited serine taxis was dependent on the carbon source utilized for growth. Further investigation of this inhibition may provide information about the mechanisms of chemotaxis in P. aeruginosa.

Amino Acids

Activation of normal genes in malignant cells: activation of chemotaxis in relation to other stages of normal differentiation in myeloid leukemia.

Genetically differerent clones of myeloid leukemic cells have been used to study the activation of normal genes in these malignant cells by the normal physiological inducer of myeloid cell differentiation, the protein MGI. In appropriate clones, MGI induced the normal differentiation-associated property of chemotaxis to a variety of compounds including the steroid hormone dexamethasone. The induced cells could also distinguish among different steroids by chemotaxis, suggesting that there are specific membrane interaction sites for steroids. The sequence of differentiation in these cells was the formation of C3 and Fc rosettes leads to phagocytosis of these rosettes and chemotaxis leads to synthesis and secretion of lysozyme leads to mature macrophages or granulocytes. The use of appropriate mutants and the comparison of induction by MGI and dexamethasone has shown that chemotaxis to casein can be dissociated from: chemotaxis to dexamethasone, ATP, and bacterial factor; formation of C3 or Fc rosettes; phagocytosis of these rosettes; synthesis of lysozyme; and the formation of mature cells. It is suggested from this dissection of normal differentiation that there are different membrane changes for specific chemotaxis, formation of these rosettes, and their phagocytosis, and that induction of each of these properties requires activation of different genes.

Animals

Defective monocyte and polymorphonuclear leukocyte chemotaxis in atopic disease.

Monocyte (MN) and polymorphonuclear (PMN) leukocyte chemotaxis was studied in 17 atopic children with hyperimmunoglobulinemia E (IgE), 9 age- and diagnosis-matched children with normal IgE levels, 10 pediatric controls, and 45 adult controls. Twenty-one of the 26 atopic patients had eczema, while 5 had only respiratory allergies. All patients were free of infection and receiving no systemic corticosteroids. Depressed PMN chemotaxis was found in only one patient. Defects of MN chemotaxis were detected in 8 of 17 atopic children with IgE and 2 of 9 with normal IgE values. Seven of 21 patients with atopic eczema and 3 of 5 with respiratory allergies had depressed MN chemoatxis. No evidence was found for a cell-directed chemotactic inhibitor in the plasma of patients with abnormal MN chemotaxis. These data demonstrate that: (1) MN chemotaxis is frequently depressed in uninfected atopic patients; (2) this abnormality occurs in patients with respiratory allergies as well as in those with eczema and is more prevalent in atopics with IgE; and (3) PMN chemotactic defects are uncommon in allergic patients. Whether abnormal MN chemotaxis is a primary or a secondary event in atopy requires further investigation.

Adolescent

Effects of tobacco smoke on chemotaxis and glucose metabolism of polymorphonuclear leukocytes.

The effect of tobacco smoke on in vitro chemotaxis of human polymorphonuclear leukocytes (PMN) was determined. Whole tobacco smoke, gas phase of smoke, and water-soluble fraction were potent inhibitors of PMN chemotaxis. The results indicated that PMN chemotaxis was inhibited in a dose-dependent manner by water-soluble fraction and that this suppression was not a result of cytotoxicity. In an attempt to determine the mechanism of chemotaxis inhibition, the effect of tobacco smoke on glucose metabolism of PMN was studied. Exposure of PMN to whole smoke, gas phase, or water-soluble fraction resulted in an increase (twofold) in glucose catabolism via both glycolysis and the hexose monophosphate shunt, with no apparent effects on the metabolism of glucose via the tricarboxylic acid cycle. These results suggest that the inhibitory effects of tobacco smoke on PMN chemotaxis were not directly attributable to effects on glucose metabolism of these cells. Further, the inhibitory effects of water-soluble fraction on PMN chemotaxis were shown to be largely irreversible and preventable in the presence of cysteine. Thus, the major inhibitory effects of tobacco smoke probably result from the direct action of oxidants and/or thiol-reactive substances on PMN.

Adult

Inhibition by cholera toxin of rat polymorphonuclear leukocyte chemotaxis demonstrated in vitro and in vivo.

The effect of cholera toxin on the chemotaxis of rat polymorphonuclear leukocytes (PMN) was studied using a technique in which the movement of the cells towards a laser-lysed erythrocyte is followed under a phase-contrast microscrope. In vitro studies indicated that the intact toxin was capable of inhibiting PMN chemotaxis in a dose-dependent manner at doses ranging from 1 to 100 ng/ml. Subunits A and B of the toxin were without inhibitory activity when used alone, but after recombination their ability to inhibit chemotaxis was similar to that of the intact toxin, suggesting that the toxin is acting intracellularly. Cholera toxin has been reported to act in other systems via stimulation of adenyl cyclase with consequent elevation of intracellular cyclic adenosine 5'-monophosphate (cAMP) levels. It appears that this mechanism may also account for its ability to inhibit chemotaxis since there was a correlation, at all doses tested, between inhibition of chemotaxis and increased intracellular cAMP levels. Cholera toxin was also found to be active in vivo in that, after intrapleural injection of the toxin, the chemotaxis of cells subsequently recovered from the pleural cavity was markedly reduced. These results support previous findings which suggest that modification of leukocyte cAMP levels can influence the chemotactic responsiveness of these cells.

Animals

Chemotaxis of Salmonella typhimurium to amino acids and some sugars.

Patterns of chemotaxis by Salmonella typhimurium strain LT-2 to l-amino acids and to several sugars were quantitated by the Adler capillary procedure. Competition experiments indicated that LT-2 possesses three predominant receptors, or interacting sets of receptors, for amino acids. These were termed the aspartate, serine, and alanine classes, respectively. Studies with strains carrying point and deletion mutations affecting components of the phosphoenolpyruvate: glycose phosphotransferase system (PTS) made unlikely a role in primary reception of d-glucose by the three soluble PTS components, namely HPr, enzyme I, and factor III. A ptsG mutant defective in membrane-bound enzyme IIB' of the high-affinity glucose transport system was shown to exhibit normal chemotaxis providing pleiotropic effects of the mutation were eliminated by its genotypic combination with other pts mutations or, phenotypically, by addition of cyclic AMP and substrate. A correlation was demonstrated between chemotaxis to glucose and activity of the low-affinity glucose transport complex, membrane-bound enzymes IIB:IIA, and an enzyme IIB:IIA mutant was shown to have a preponderant defect in chemotaxis to glucose and mannose. Of four systems capable of galactose transport, only the beta-methylgalactoside transport system was implicated in chemotaxis to galactose. Some properties of a mutant possibly defective in processing of signals for chemotaxis to sugars is described.

Amino Acids

Clinical conditions associated with defective polymorphonuclear leukocyte chemotaxis.

Impressive numbers of clinical conditions are associated with defective leukocyte chemotaxis. In many, this cellular dysfunction is associated with other abnormalities of the immune response, but in others abnormal chemotactic responsiveness of leukocytes is the only abnormality of function identified in the laboratory. Patients are usually selected for study because of unusually severe, recurrent infections or poor response to antimicrobial agents, and therefore a frequent association between abnormality of chemotaxis and infection would be expected. Many patients demonstrate abnormal chemotaxis during remissions as well as during infections, and there seems little doubt that abnormality of chemotaxis is related to susceptibility to infections. Partial classification of disorders of chemotaxis was attempted. Major abnormalities are found when there is a primary cellular disorder or cell-directed inhibitors of chemotaxis are found. Less marked abnormalities are found when chemotactic factors are deficient.

Bacterial Infections

Defective monocyte chemotaxis in mycosis fungoides: lack of essential helper lymphocytes.

The effect of lymphocytes on monocytes chemotaxis in vitro was studied using lymphocyte fractions prepared by glass bead column separation and mononuclear cell fractions prepared by Ficoll-Hypaque separation. The diminished monocyte chemotaxis in ten patients with mycosis fungoides was corrected in vitro by the addition of normal lymphocytes. This helper effect was not mediated by soluble factors. Lymphocytes from mycosis fungoides patients did not inhibit chemotaxis by monocytes from mycosis fungoides patients did not inhibit chemotaxis by monocytes from normal donors. No cell directed chemotactic inhibitor, differences in LDCF production in vitro or differences in circulating chemattractants were found. These data support the conclusion that the abnormal monocyte chemotaxis seen in mycosis fungoides is due to lack of essential helper lymphocyte function and represents an abnormal mononuclear cell interaction which may be important in the establishment of the mononuclear cell infiltrate in mycosis fungoides.

Adult

Localization of submembranous cations to the leading end of human neutrophils during chemotaxis.

Potassium pyroantimonate was used to localize sites of bound cations in human neutrophils under conditions of random migration, stimulated random migration (chemokinesis), and directed migration (chemotaxis). The cells were placed in a standard chamber in which 0.45-micron micropore filters separated the cells from the stimulus (buffer, Escherichia coli endotoxin-activated serum or the synthetic chemotactic peptide N-formyl-Met-Leu-Phe). The small pore filters permitted pseudopod formation but impeded cell imgration through the filter. Cells examined under all conditions had electron-dense precipitates of antimonate salts in some granules. However, antimonate deposits were localized in the condensed chromatin of the nucleus during random migration and associated to a large extent with the uncondensed nuclear chromatin during chemokinesis and chemotaxis. Under conditions of chemokinesis deposition of antimonate procipitates appeared on the cytoplasmic side of the plasma membrane of neutrophils whereas under conditions of chemotaxis cation deposits beneath the cell membrane were localized to the pseudopods which were directed toward the chemoattractant. In addition to endotoxin-activated serum, concentrations of N-formyl-Met-Leu-Phe which caused neutrophil chemotaxis (10(-8) M) also caused cation deposition beneath the cell membrane at the leading end of the cell regardless of whether albumin was present in the incubation media. However, with higher concentrations of the synthetic peptide (10(-5) M) which caused granule release and were not chemotactic, submembranous cation deposition was not seen. EDTA (10 mM) and EGTA (10 mM) removed nuclear, granular, and submembranous cation deposits from neutrophils examined under conditions of chemotaxis. X-ray microprobe analysis of antimonate deposits revealed the possible presence of calcium but did not detect sodium or magnesium. The data indicate that chemotactic factors induce submembranous deposition of cations, most likely Ca++, which localize to the leading edge of cells exposed to a gradient of chemoattractant.

Antimony

Effect of morphine analogues on chemotaxis in Escherichia coli.

Pretreatment of Escherichia coli w3110 with levorphanol, a morphine analogue, reduced chemotaxis to serine, aspartic acid and galactose. This decreased chemotaxis was not due to decreased viability or motility. Pretreatment with 1.1 mM-levorphanol for 1 h, followed by washing to remove the drug prior to determination of chemotaxis, inhibited chemotaxis to each of the attractants by at least 80%. Pretreatment with dextrorphan, the enantiomorph of levorphanol, or levallorphan, the N-allyl analogue of levorphanol, resulted in a similar inhibition of chemotaxis. Reversal of the inhibition produced by pretreatment with levorphanol required a period of growth of at least one generation time.

Aspartic Acid

Mononuclear leucocyte chemotaxis in Boyden chambers: inhibition by subantimitotic concentrations of antitubulins.

The chemotaxis of Lymphoprep -isolated human mononuclear leucocytes (L-MNs) from peripheral blood was inhibited by subantimitotic concentrations of the antitubulins demecolcine, podophyllic acid ethylhydrazide, vinblastine and griseofulvin. It is suggest that L-MN chemotaxis, like polymorphonuclear leucocyte (PMN) chemotaxis, is composed of a direct antitubulin-insensitive chemotaxis and a leukocyte-induced antitubulin-sensitive chemotaxis.

Antineoplastic Agents