Chemotaxis or migration inhibition of rabbit peritoneal polymorphonuclear leukocytes caused by chemoattractants at various concentrations

Purified lipopolysaccharide (LPS) from Veillonella incubated in normal rabbit serum was tested for chemotactic activity on rabbit polymorphonuclear leukocytes (PMNs) in modified Boyden chambers. In doses above those giving optimal response (over-optimal dose), a decrease of the PMN migration activity was found. This decrease also correlated well with an increase in the migration inhibition of the PMNs as demonstrated with the capillary tube assay. The PMN chemotactic factor isolated from LPS-induced inflammatory exudate (LPS-CF) in rabbits, produced both a decrease in chemotactic response and a migration inhibition of PMNs in over-optimal doses. This inhibitory effect was not due to cytotoxicity, proved by the trypan blue exclusion test. Also, a reduced locomotion of PMNs first preincubated with chemoattractants and then reactivated, was shown when the same PMNs were restimulated to migration using the same chemoattractants. This was interpreted as a deactivation of the cells. A cross-deactivation was demonstrated between LPS-CF and casein. The results from the experiments reported show that the Boyden chamber may be used to disciminate directional chemotaxis and migration inhibition. It may also be concluded from the study that the reduced migration activity of PMNs at over-optimal doses of chemoattractants is not due to cytotoxicity, but most probably is caused by a deactivation of the cells.     

The difference between random movement and chemotaxis. Effects of antitubulins on neutrophil granulocyte locomotion

The antitubulins demecolcine and podophyllic acid ethylhydrazide (SPI) were used in experiments designed to elucidate the role of centriole-associated cytoplasmic microtubules in the locomotion of human neutrophil granulocytes (PMNs). The PMN locomotion was studied as chemotaxis and as the velocity of random movement. The PMN chemotaxis was inhibited by demecolcine (0.01 μg/ml) and SPI (0.1 μg/ml), i.e. concentrations below the reported threshold ones for mitotic arrest in metaphase. The velocity of single PMNs during random movement was only slightly reduced by treatment with 0.5 μg/ml of SPI. PMN locomotion was not appreciably inhibited by SPI, 0.5 μg/ml. The discrepancies mentioned suggest that centriole-associated microtubules are essential structures in the PMN direction-finding or PMN directional movement of chemotaxis but not in the mechanism of PMN locomotion. The present observations might, at least in part, explain the beneficial effects of antitubulins on acute gout.     

Developmental changes in chemotactic response and choice of two attractants, sodium acetate and diacetyl, in the nematode Caenorhabditis elegans

The chemotactic behavior of the nematode Caenorhabditis elegans to chemical attractants, water-soluble sodium acetate and odorant diacetyl, was investigated using nematodes at various developmental stages to examine the effects of postembryonic development on chemotactic response and spontaneous locomotion. The chemotactic responses to attractants increased as development progressed, and the largest responses to either 1.0 M sodium acetate or 0.1% diacetyl were seen at the young adult (YA) or day adult (A1) stage, respectively. Responses to the chemicals declined thereafter in-line with increasing age. The chemotaxis indices for attractants correlated with activity of spontaneous locomotion (p<0.01), suggesting that a change in spontaneous locomotion is one of the factors involved with the change in chemotactic responses during development. We also investigated the effect of aging on attractant choice by the simultaneous presentation of 0.6 M sodium acetate and 0.1% diacetyl. In the presence of both attractants, the fraction of larval animals at the sodium acetate location was greater than that at the diacetyl location (p<0.05). The fractions of YA animals that gathered at either location were almost identical, whereas the fraction of adult animals at the diacetyl location was greater than that at the sodium acetate location (p<0.05). The patterns of attractant choice of the long-lived daf-2 mutants and short lifespan mev-1 mutants showed the same tendency as those of wild type nematodes in the presence of both attractants. These results suggest that a change in the neuronal mechanisms controlling attractant choice and preference occurs during developmental progression.     

Human polymorphonuclear leukocytes respond to waves of chemoattractant,like Dictyostelium

It has been assumed that the natural chemotactic signal that attracts human polymorphonuclear leukocytes (PMNs) over long distances to sites of infection is in the form of a standing spatial gradient of chemoattractant. We have questioned this assumption on the grounds, first, that standing spatial gradients may not be stable over long distances for long periods of time and, second, that in the one animal cell chemotaxis system in which the natural chemotactic signal has been described in space and time, aggregation of Dicytostelium discoideum, the signal is in the form of an outwardly relayed, nondissipating wave of attractant. Here, it is demonstrated that PMNs alter their behavior in each of the four phases of a wave of PMN chemoattractant, fashioned after the Dictyostelium wave, in a manner similar to Dictyostelium. These results demonstrate that PMNs have all of the machinery to respond to a natural wave of attractant, providing support to the hypothesis that the natural signal that attracts PMNs over large distances to sites of infection in the human body may also be in the form of a wave.     

How do leucocytes perceive chemical gradients?

Abstract Chemoattractants determine not only the direction of leucocyte locomotion (chemotaxis) but also its speed (chemokinesis). Various mechanisms by which leucocytes may detect chemotactic gradients, including spatial and temporal detection, are briefly reviewed. These mechanisms as originally proposed did not address the question how attractants cause leucocytes to migrate in persistent random paths in the absence of a gradient. Stochastic models have recently been presented in which leucocytes either respond by polarizing and migrating in the direction from which they receive their first signal, or respond to random flucuations in the perceived attractant concentration. Stochastic models allow an explanation for the persistent random walk shown by cells in uniform concentrations of attractant as well as for directional locomotion in gradients. They suggest that, at the biochemical level, the mechanisms by which attractants stimulate chemotaxis and chemokinesis are probably the same.     

How do leucocytes perceive chemical gradients?

Chemoattractants determine not only the direction of leucocyte locomotion (chemotaxis) but also its speed (chemokinesis). Various mechanisms by which leucocytes may detect chemotactic gradients, including spatial and temporal detection, are briefly reviewed. These mechanisms as originally proposed did not address the question how attractants cause leucocytes to migrate in persistent random paths in the absence of a gradient. Stochastic models have recently been presented in which leucocytes either respond by polarizing and migrating in the direction from which they receive their first signal, or respond to random fluctuations in the perceived attractant concentration. Stochastic models allow an explanation for the persistent random walk shown by cells in uniform concentrations of attractant as well as for directional locomotion in gradients. They suggest that, at the biochemical level, the mechanisms by which attractants stimulate chemotaxis and chemokinesis are probably the same.     

Ability of polymorphonuclear leukocytes to orient in gradients of chemotactic factors

Polymorphonuclear leukocyte (PMN) chemotaxis has been examined under conditions which allow phase microscope observations of cells responding to controlled gradients of chemotactic factors. With this visual assay, PMNs can be seen to orient rapidly and reversibly to gradients of N-formylmethionyl peptides. The level of orientation depends upon the mean concentration of peptide present as well as the concentration gradient. The response allows an estimation of the binding constant of the peptide to the cell. In optimal gradients, PMNs can detect a 1% difference in the concentration of peptide. At high cell densities, PMNs incubated with active peptides orient their locomotion away from the center of the cell population. This orientation appears to be due to inactivation of the peptides by the cells. Such inactivation in vivo could help to limit an inflammatory response.     

Amplification of the activity of human leukocyte inhibitory factor (LIF) by the generation of a low molecular weight inhibitor of PMN leukocyte chemotaxis

Leukocyte inhibitory factor (LIF), which was derived from human peripheral blood lymphocytes by stimulation with concanavalin A ad partially purified by Sephadex G-100 gel filtration, inhibited the in vitro spontaneous migration and chemotaxis of human PMN leukocytes as assessed in a Boyden chamber micropore filter assay. The inhibitory activity was attributed to LIF, a principle defined in terms of its inhibition of PMN leukocyte migration from glass capillary tubes since it was preferentially directed to PMN leukocytes as compared to mononuclear leukocytes, exhibited a size comparable to LIF by gel filtration, and was inactivated by diisopropyl fluorophosphate in parallel with LIF. Incubation of PMN leukocytes with LIF released additional inhibitory activity, distinct from LIF, which resembled the neutrophil-immobilizing factor (NIF) by virtue of its approximate m.w. of 4000 by filtration on Sephadex G-25, inactivation by trypsin digestion, and preferential noncytotoxic inhibition of spontaneous migration and chemotaxis of PMN leukocytes as compared to mononuclear leukocytes. Thus LIF inhibits PMN leukocyte migration both by a direct action on the cells and by an amplification pathway that is mediated by low m.w. chemotactic inhibitors similar to NIF.     

Thrombospondin stimulates motility of human neutrophils

Polymorphonuclear leukocytes (PMNs) migrate to sites of inflammation or injury in response to chemoattractants released at those sites. The presence of extracellular matrix (ECM) proteins at these sites may influence PMN accumulation at blood vessel walls and enhance their ability to move through tissue. Thrombospondin (TSP), a 450-kD ECM protein whose major proteolytic fragments are a COOH-terminal 140-kD fragment and an NH2-terminal heparin-binding domain (HBD), is secreted by platelets, endothelial cells, and smooth muscle cells. TSP binds specifically to PMN surface receptors and has been shown, in other cell types, to promote directed movement. TSP in solution at low concentrations (30-50 nM) "primed" PMNs for f-Met-Leu-Phe (fMLP)- mediated chemotaxis, increasing the response two- to fourfold. A monoclonal antibody against the HBD of TSP totally abolished this priming effect suggesting that the priming activity resides in the HBD of TSP. Purified HBD retains the priming activity of TSP thereby corroborating the antibody data. TSP alone, in solution at high concentrations (0.5-3.0 microM), stimulated chemotaxis of PMNs and required both the HBD and the 140-kD fragment of TSP. In contrast to TSP in solution, TSP bound to nitrocellulose filters in the range of 20- 70 pmol stimulated random locomotion of PMNs. The number of PMNs migrating in response to bound TSP was approximately two orders of magnitude greater than the number of cells that exhibited chemotaxis in response to soluble TSP or fMLP. Monoclonal antibody C6.7, which recognizes an epitope near the carboxyl terminus of TSP, blocked migration stimulated by bound TSP, suggesting that the activity resides in this domain. Using proteolytic fragments, we demonstrated that bound 140-kD fragment, but not HBD, promoted migration of PMNs. Therefore, TSP released at injury sites, alone or in synergy with chemotactic peptides like fMLP, could play a role in directing PMN movement.     

Involvement of transport in Rhodobacter sphaeroides chemotaxis.

The chemotactic response to a range of chemicals was investigated in the photosynthetic bacterium Rhodobacter sphaeroides, an organism known to lack conventional methyl-accepting sensory transduction proteins. Strong attractants included monocarboxylic acids and monovalent cations. Results suggest that the chemotactic response required the uptake of the chemoeffector, but not its metabolism. If a chemoeffector could block the uptake of another attractant, it also inhibited chemotaxis to that attractant. Sodium benzoate was not an attractant but was a competitive inhibitor of the propionate uptake system. Binding in an active uptake system was therefore insufficient to cause a chemotactic response. At different concentrations, benzoate either blocked propionate chemotaxis or reduced the sensitivity of propionate chemotaxis, an effect consistent with its role as a competitive inhibitor of uptake. Bacteria only showed chemotaxis to ammonium when grown under ammonia-limited conditions, which derepressed the ammonium transport system. Both chemotaxis and uptake were sensitive to the proton ionophore carbonyl cyanide m-chlorophenylhydrazone, suggesting an involvement of the proton motive force in chemotaxis, at least at the level of transport. There was no evidence for internal pH as a sensory signal. These results suggest a requirement for the uptake of attractants in chemotactic sensing in R. sphaeroides.     

Analysis of polymorphonuclear leukocyte (PMN) migration by the leading-front technique : Random locomotion and chemotaxis

The present study was designed to elucidate the contribution of non-stimulated random movement, stimulated random movement, antitubulin-resistant chemotaxis and antitubulin-sensitive chemotaxis to the casein-induced PMN migration into a micropore filter, evaluated by the leading-front technique. This analysis was conducted by a simplified test design including PMN migration, (a) without casein; (b) in a gradient of casein; and (c) in casein without gradient. Treatment with the antitubulin SPI (a podophyllotoxin derivative) inhibited PMN migration within a casein gradient down to the level of the stimulated PMN random movement induced by casein. The casein-induced PMN chemotaxis measured by the leading-front technique is thus composed of stimulated random movement and antitubulin-sensitive chemotaxis without evidence of antitubulin-resistant chemotaxis. It is suggested that the anti-inflammatory effects of the antitubulins (colchicine, podophyllotoxin, Vinca alcaloids, griseofulvin) are due to an inhibition of the antitubulin-sensitive chemotaxis.     

Linkage between blood coagulation and inflammation: stimulation of neutrophil tissue kallikrein by thrombin

There has been major interest in the potential interaction between blood coagulation and inflammation. Most of the effort has focused on cellular interactions involving platelets and polymorphonuclear leukocytes (PMNS). The recent discovery of tissue kallikrein(TK) activity in PMNs prompted the study of the possible role of thrombin(IIa) in this process. Human PMNs were isolated by density gradient centrifugation. Human IIa was compared with fMLP with respect to chemotaxis and enzyme release. Results from the challenges by IIa and fMLP were compared to a NaCl control using Student's paired t-test. IIa was a potent chemotactic agent for PMNs (p less than or equal to 0.0121) and stimulated the release of TK (p less than or equal to 0.0001) as determined by hydrolysis of S-2266. FMLP significantly stimulated PMN chemotaxis (p less than or equal to 0.0028) but had no effect on TK release. Release of TK was confirmed by Western Blot analysis and 35S-methionine incorporation into a 35 KD protein after IIa challenge. These results demonstrate that IIa is chemotactic for PMNs and can cause release of tissue kallikrein demonstrating a direct role for blood coagulation in the regulation of the inflammatory response.     

Inflammatory process caused by intraperitoneal injection of polyester in the rat: chemotactic activity in lavage fluid from the cavity

Minced polyester threads introduced into peritoneal cavity of guinea pigs or rats cause a granulomatous inflammation with evidence of macrophage stimulation. Chemotactic agents play an important role in the inflammatory reaction; they may be exogenous and/or endogenous. These are released locally by the cells involved in inflammation. In this paper the chemotactic effects of the peritoneal fluids from rats bearing the polyester inflammatory process, have been studied on PMN cells "in vitro". The peritoneal cavity fluids were obtained by washing the cavity of untreated rats or rats intraperitoneally injected with polyester, 1, 3, 7, 14 days after the intraperitoneal injection. The chemotactic response was assayed by employing modified chemotaxis Boyden chambers (Blind Well Neuro Probe) and polymorphonuclear leukocytes from normal or treated rats. Quantification of the migration was calculated by chemotactic index (A/B) (B = random migration, A = chemotaxis). The results demonstrated that the peritoneal fluids taken 3 and 7 days after the intraperitoneal polyester injection, elicit an evident chemotaxis response greater than that showed by peritoneal fluids from control rats. It is suggested that chemotactic factors can be produced and released by mononuclear cells involved in the inflammatory process.     

The effects of platelet activating factor and retinoic acid on the expression of ELAM-1 and ICAM-1 and the functions of neutrophils

Preincubation of pulmonary microvascular endothelial cells (PMVECs) with platelet-activating factor (PAF) for 3.5 h increased the adhesion rate of polymorphonuclear leukocytes (PMNs) to PMVECs from 57.3% to 72.8% (p < 0.01). Preincubation of PMNs with PAF also increased PMN-PMVEC adhesion rate. All-trans retinoic acid (RA) blocked the adherence of untreated PMNs to PAF-pretreated PMVECs but not the adherence of PAF-pretreated PMNs to untreated PMVECs. PAF increased the expression of intercellular adhesion molecule-1 (ICAM-1) and E-selection (ELAM-1) on PMVECs, PMN chemotaxis to zymosan-activated serum and histamine, and PMN aggregation and the release of acid phosphatase from PMNs. Co-incubation of RA inhibited PAF-induced PMN aggregation, the release of acid phosphatase from PMNs, and PMN chemotaxis to zymosan-activated serum and histamine while the expression of ICAM-1 and ELAM-1 did not change. Our results suggest that RA can be used to ameliorate PMN-mediated inflammation.     

Effect of temperature on Pseudomonas fluorescens chemotaxis.

The effects of temperature and attractants on chemotaxis in psychrotrophic Pseudomonas fluorescens were examined using the Adler capillary assay technique. Several organic acids, amino acids, and uronic acids were shown to be attractants, whereas glucose and its oxidation products, gluconate and 2-ketogluconate, elicited no detectable response. Chemotaxis toward many attractants was dependent on prior growth of the microorganism with these compounds. However, the organic acids, malate and succinate, caused strong chemotactic responses regardless of the carbon source used for growth of the bacteria. The temperature at which the cells were grown (30 or 5 degrees C) had no significant detectable effect on chemotaxis to the above attractants. The temperature at which the cells were assayed appeared to affect the rate but the extent of the chemotactic response, nor the concentration response curves. The ratios of the rate of accumulation of cells to the attractant malate were approximately 2, 4, and 1 at 30, 17, and 5 degrees C, respectively. Strong chemotactic responses were observed with cells assayed at temperatures approaching 0 degree C and appeared to be functional over a broad temperature range of 3 to 35 degrees C.     

Interactions between Caenorhabditis elegans individuals during chemotactic response

The chemotactic response of the nematode Caenorhabditis elegans is known to be affected by the population density on an assay plate, suggesting the existence of interactions between individual animals. To clarify the interactions between individuals during chemotaxis, we investigated the effect of population density at an attractant area on the chemotactic response to water-soluble sodium acetate and odorant diacetyl using wild-type N2 animals and daf-22 (m130) mutants, which have defective pheromone secretion but can sense pheromone. The chemotaxis index of N2 animals at 90 min of the assay negatively correlated with the number of animals on the assay plate regardless of the type of attractant used (p<0.01). On the other hand, there was no significant difference in the chemotaxis indices of daf-22 (m130) mutants for either of the attractants between the low-and high-population groups. When daf-22 (m130) mutants of a high population density were placed at the attractant location in advance, the chemotaxis index of N2 animals was almost the same as that in the control assay in which no animals were placed at the attractant location in advance. When N2 animals of a high population density were placed at the attractant location in advance, the chemotaxis indices of N2 animals and daf-22 (m130) mutants were significantly smaller than those obtained in the control assay (p<0.05). In the absence of an attractant, we observed a decline in the fraction of animals in the neighborhood of N2 animals of a high population density, although the nematodes were not influenced by daf-22 (m130) mutants of a high population density. These results suggest that the attraction of nematodes to chemicals is inhibited by an increase in the concentration of the pheromone generated by N2 animals at the attractant location.     

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.     

Recombinant human tumor necrosis factor alpha lacks chemotactic activity for human peripheral blood neutrophils and monocytes

Preparations of recombinant human tumor necrosis factor alpha (rhuTNF alpha) free of aminoterminal methionine were tested for human neutrophil granulocyte (PMN) and monocyte (MO) chemotactic activity using the Boyden chamber system. Over a wide range of concentrations (10(-7)-10(-15) M) rhuTNF alpha of two different sources failed to elicit chemotactic responses in PMN or MO, whereas strong PMN and MO chemotactic activity could be detected using the tripeptide N-formyl-methionyl-leucyl-phenylalanine (FMLP). In addition, rhuTNF alpha containing 62% aminoterminal methionine failed to induce PMN and MO chemotaxis. It is concluded that rhuTNF alpha may not be a chemotaxin for human PMN and MO in vitro.     

Chemotactic response of Escherichia coli to chemically synthesized amino acids.

In Escherichia coli, seven of the commonly occurring amino acids are strong attractants: L-aspartate, L-serine, L-glutamate, L-alanine, L-asparagine, glycine, and L-cysteine, in order of decreasing effectiveness. The chemotactic response to each amino acid attractant is mediated by either methyl-accepting chemotaxis protein I or II, but not by both. Seven of the commonly occurring amino acids are repellents. This work was carried out with chemically synthesized amino acids.     

ClC-3 and IClswell are required for normal neutrophil chemotaxis and shape change

Polymorphonuclear leukocytes undergo directed movement to sites of infection, a complex process known as chemotaxis. Extension of the polymorphonuclear leukocyte (PMN) leading edge toward a chemoattractant in association with uropod retraction must involve a coordinated increase/decrease in membrane, redistribution of cell volume, or both. Deficits in PMN phagocytosis and trans-endothelial migration, both highly motile PMN functions, suggested that the anion transporters, ClC-3 and ICl(swell), are involved in cell motility and shape change ( Moreland, J. G., Davis, A. P., Bailey, G., Nauseef, W. M., and Lamb, F. S. (2006) J. Biol. Chem. 281, 12277-12288 ). We hypothesized that ClC-3 and ICl(swell) are required for normal PMN chemotaxis through regulation of cell volume and shape change. Using complementary chemotaxis assays, EZ-TAXIScantrade mark and dynamic imaging analysis software, we analyzed the directed cell movement and morphology of PMNs lacking normal anion transporter function. Murine Clcn3(-/-) PMNs and human PMNs treated with anion transporter inhibitors demonstrated impaired chemotaxis in response to formyl peptide. This included decreased cell velocity and failure to undergo normal cycles of elongation and retraction. Impaired chemotaxis was not due to a diminished number of formyl peptide receptors in either murine or human PMNs, as measured by flow cytometry. Murine Clcn3(-/-) and Clcn3(+/+) PMNs demonstrated a similar regulatory volume decrease, indicating that the ICl(swell) response to hypotonic challenge was intact in these cells. We further demonstrated that ICl(swell) is essential for shape change during human PMN chemotaxis. We speculate that ClC-3 and ICl(swell) have unique roles in regulation of PMN chemotaxis; ICl(swell) through direct effects on PMN volume and ClC-3 through regulation of ICl(swell).