Chemotactic Activity For Polymorphonuclear Leukocytes Research Articles

The human antimicrobial and chemotactic peptides LL-37 and α-defensins are expressed by specific lymphocyte and monocyte populations

We identified antibacterial components in human T and natural killer (NK) cells by using freshly isolated lymphocytes enriched for T and NK cells as starting material. After growing these lymphocytes for 5 days in the presence of interleukin (IL)–2, we isolated and characterized several antibacterial peptides/proteins from the supernatant—α-defensins (HNP 1-3), LL-37, lysozyme, and a fragment of histone H2B—although other active components were also present. We then used reverse transcriptase–polymerase chain reaction to search for expression of the gene coding for LL-37 in several B-cell lines, γδ T-cell lines, NK clones, and one monocytic cell line, with positive results, but found no expression in several αβ T-cell lines. The α-defensins (HNP 1-3) were also found to be expressed in several of these cell lines. To confirm the presence of these antibacterial peptides in lymphocytes, we localized them to NK, γδ T cells, B cells, and monocytes/macrophages by using double-staining immunohistochemical analysis of freshly isolated lymphocytes. We also found that primary cultures of lymphocytes transcribe and secrete LL-37 and that these processes are affected by IL-6 and interferon-γ. In addition, we demonstrated that LL-37 has chemotactic activity for polymorphonuclear leukocytes and CD4 T lymphocytes, whereas others have shown chemotactic activity for human α-defensins (HNP 1-2). These findings suggest that microbicidal peptides are effector molecules of lymphocytes and that antibacterial activity previously shown to be derived from T and NK cells may be partly mediated by the antibacterial peptides LL-37 and HNP 1-3.

Chemotactic activity for polymorphonuclear leukocytes: meconium versus meconium-stained amniotic fluid.

Our previous study indicated that meconium-stained amniotic fluid (turbid AF) possessed a potent chemotactic activity for leukocytes, which may be dependent on interleukin-8. It is not known, however, whether meconium itself possesses this chemotactic activity. Meconium samples were collected from mature neonates with and without turbid AF. A 5% meconium suspension in phosphate buffered saline was prepared and measured for its chemotactic activity for leukocytes using the blind well chamber technique. Concentrations of IL-8, TNFalpha and IL-1beta were also measured with ELISA. The number of leukocytes that migrated to the meconium suspension (35 +/- 27) was comparable with that of the clear AF (31 +/- 37), but was significantly lower than that of the turbid AF (184 +/- 62, P < 0.0001). The meconium suspension contained much lower levels of IL-8, TNFalpha and IL-1beta than the turbid AF. Meconium itself exhibits a lower chemotactic activity for polymorphonuclear leukocytes than turbid AF in vitro. The leukocyte chemotactic activity of turbid AF does not originate from meconium itself.

The human antimicrobial and chemotactic peptides LL-37 and α-defensins are expressed by specific lymphocyte and monocyte populations

AbstractWe identified antibacterial components in human T and natural killer (NK) cells by using freshly isolated lymphocytes enriched for T and NK cells as starting material. After growing these lymphocytes for 5 days in the presence of interleukin (IL)–2, we isolated and characterized several antibacterial peptides/proteins from the supernatant—α-defensins (HNP 1-3), LL-37, lysozyme, and a fragment of histone H2B—although other active components were also present. We then used reverse transcriptase–polymerase chain reaction to search for expression of the gene coding for LL-37 in several B-cell lines, γδ T-cell lines, NK clones, and one monocytic cell line, with positive results, but found no expression in several αβ T-cell lines. The α-defensins (HNP 1-3) were also found to be expressed in several of these cell lines. To confirm the presence of these antibacterial peptides in lymphocytes, we localized them to NK, γδ T cells, B cells, and monocytes/macrophages by using double-staining immunohistochemical analysis of freshly isolated lymphocytes. We also found that primary cultures of lymphocytes transcribe and secrete LL-37 and that these processes are affected by IL-6 and interferon-γ. In addition, we demonstrated that LL-37 has chemotactic activity for polymorphonuclear leukocytes and CD4 T lymphocytes, whereas others have shown chemotactic activity for human α-defensins (HNP 1-2). These findings suggest that microbicidal peptides are effector molecules of lymphocytes and that antibacterial activity previously shown to be derived from T and NK cells may be partly mediated by the antibacterial peptides LL-37 and HNP 1-3.

Meconium-stained amniotic fluid exhibits chemotactic activity for polymorphonuclear leukocytes in vitro

We have studied whether meconium-stained, turbid amniotic fluid (turbid AF) obtained during term pregnancy possesses chemotactic activity for polymorphonuclear leukocytes (PMNs) in the absence of clinically apparent infection. Eight samples of turbid AF were obtained from eight women who underwent a cesarean section (four emergency and four elective cesarean sections) in the absence of signs of clinical infection or fetal distress. Samples of clear AF obtained from nine women during an elective cesarean section served as a control. We used also a negative control (medium only) and a positive control containing 10 nM N-formyl-methionyl-leucyl-phenylalanine. The control or turbid AF specimen was placed in the lower compartment of a blind well chamber, and the PMN suspension was placed in the upper compartment. Following incubation, the number of PMNs that had migrated and passed through the filter to the AF was counted. The number of control PMNs that migrated to the turbid AF (200±59) was comparable to that of the positive (162±24) but significantly exceeded that of the clear AF (17±11; P<0.0001) and of the negative control (25±9; P<0.0001). Checkerboard assay indicated that the turbid AF exhibited a dose-dependent chemotactic activity for PMNs. The turbid AF contained higher levels of TNFα, IL-1β and IL-8 than the clear AF. The concentration of IL-8 in the AF was correlated positively with the chemotactic activity of the AF ( r=0.733, P=0.0005). Anti-human IL-8 antibody added in the turbid AF dose-dependently abolished the chemotactic activity of the turbid AF. It is concluded that meconium-stained AF is a chemoattractant for PMNs and that cytokines such as an IL-8 may be involved in this phenomenon.

Meconium-stained amniotic fluid exhibits chemotactic activity for polymorphonuclear leukocytes in vitro

International Journal of Gynecology & ObstetricsVolume 70, Issue S2 p. B157-B158 Pre-term birth Meconium-stained amniotic fluid exhibits chemotactic activity for polymorphonuclear leukocytes in vitro T. Yamada, T. YamadaSearch for more papers by this authorH. Minakami, H. MinakamiSearch for more papers by this authorS. Matsubara, S. MatsubaraSearch for more papers by this authorY. Kohmura, Y. KohmuraSearch for more papers by this authorM. Hiratsuka, M. HiratsukaSearch for more papers by this authorI. Sato, I. SatoSearch for more papers by this author T. Yamada, T. YamadaSearch for more papers by this authorH. Minakami, H. MinakamiSearch for more papers by this authorS. Matsubara, S. MatsubaraSearch for more papers by this authorY. Kohmura, Y. KohmuraSearch for more papers by this authorM. Hiratsuka, M. HiratsukaSearch for more papers by this authorI. Sato, I. SatoSearch for more papers by this author First published: 10 December 2003 https://doi.org/10.1016/S0020-7292(00)83236-1AboutPDF ToolsRequest permissionExport citationAdd to favoritesTrack citation ShareShare Give accessShare full text accessShare full-text accessPlease review our Terms and Conditions of Use and check box below to share full-text version of article.I have read and accept the Wiley Online Library Terms and Conditions of UseShareable LinkUse the link below to share a full-text version of this article with your friends and colleagues. Learn more.Copy URL Share a linkShare onFacebookTwitterLinked InRedditWechat No abstract is available for this article. Volume70, IssueS22000Pages B157-B158 RelatedInformation

Chemotactic activity and IL-8 levels in the cerebrospinal fluid in canine steroid responsive meningitis–arteriitis

Steroid responsive meningitis–arteriitis (SRMA) is a systemic immune disorder, characterized by inflammatory-stenosing lesions of the meningeal arteries and meningitis. The predilection of the disease for the central nervous system (CNS) remains unexplained. In this study, chemotactic activity and chemotactic factors were measured in the cerebrospinal fluid (CSF) of dogs with SRMA. CSF of dogs with SRMA exerted a marked chemotactic activity for leukocytes. Neutrophils were attracted to a similar degree as by CSF from animals with bacterial encephalitis. Chemotactic activity was also noted for mononuclear cells, however, by far weaker than in CSF from animals with viral encephalitis. While the inflammatory process could be suppressed with glucocorticoid treatment, the chemotactic activity of CSF persisted. We could identify IL-8-like activities using a desensitization assay in the CSF of animals with SRMA and also found increased IgA levels. Increased chemotactic activity for polymorphonuclear leukocytes correlated positively with the levels of IL-8-like activity in CSF. Our observations clearly suggest that in SRMA chemotactic factors are generated in the CNS. These include IL-8, but probably also others. The intensity of this production appears to correlate with IgA levels in the CSF suggesting either a causal link or reflecting the severity of the inflammation.

Cellular immuno-competence of infected root canal contents in pathogenesis of periapical lesions

The soluble fractions of infected root canal contents (IRCC) were collected from about 300 human extracted teeth and examined for the presence of mononuclear cell (MNC) chemotaxis and cellular immunocompetence. IRCC showed remarkable chemotactic activity for polymorphonuclear leukocytes but a weak activity for MNC. However, generation of intrinsic MNC chemotaxis and induction of cellular immunity were confirmed in rats given repeated injections of IRCC.

Influence of recombinant human erythropoietin therapy on in vivo chemotaxis and in vitro phagocytosis of polymorphonuclear cells of hemodialysis patients.

Recombinant human erythropoietin (rhu-EPO) mainly stimulates erythropoiesis, but also influences the production and function of polymorphonuclear granulocytes (PAN). We evaluated the influence of rhu-EPO therapy in patients on maintenance hemodialysis without iron overload on both phagocytic and chemotactic activity of polymorphonuclear leukocytes (PMN) obtained from blood and a skin chamber. The study comprised 28 dialysis patients (15 females, 13 males, age 22-78 years, dialysis sessions three times weekly 4-5 h, mean duration of dialysis treatment 4-99 months) and 20 healthy subjects (9 females, 11 males, age 18-56 years). The absolute number of leukocytes in the peripheral blood, the leukocyte mobilization (chemotactic activity) and the phagocytic activity of PMNs were tested in patients before (n = 28), and after (n = 18) reaching the target hematocrit of 30% and during the follow-up period. No alteration in the total leukocyte number (PMN, monocytes, lymphocytes) in the peripheral blood during rhu-EPO therapy could be seen. The phagocytic activity of PMNs slightly improved during the follow-up period whereas the decreased chemotactic activity remained unchanged.

Preliminary characterization of Pseudomonas aeruginosa peptide chemotactins for polymorphonuclear leukocytes.

In a previous report, we showed that supernatants of Pseudomonas aeruginosa cultures exhibit chemotactic activity for polymorphonuclear leukocytes (PMNL). In this study, P. aeruginosa chemotactins were isolated, purified, and partially characterized. The organisms were cultured in Vogel-Bonner defined medium, and cultures were stopped in late log phase. Chemotactins withstood heating, remained unaltered after acid or alkali treatment in a pH range from 4 to 10, and resisted digestion by trypsin or carboxypeptidase, but chemotactic activity was decreased by 73% after incubation with pronase. Only 2% of the total chemotactic activity of culture supernatants could be extracted with chloroform. Chemotactins with molecular sizes less than 3 kDa constituted the largest contribution to the chemotactic activity of culture supernatants. Pretreatment of PMNL with 10(-5) M formylmethionyl-leucyl-phenylalanine (FMLP) inhibited chemotaxis towards FMLP and P. aeruginosa culture supernatants but not towards complement component C5a. In conclusion, the total chemotactic activity for PMNL of P. aeruginosa culture supernatants was due, almost exclusively, to chemotactins that have properties similar, if not identical, to those exhibited by formylmethionyl peptides.

Neutrophilic alveolitis in idiopathic pulmonary fibrosis. The role of interleukin-8.

Idiopathic pulmonary fibrosis is an immunologically mediated pulmonary disorder in which activated alveolar macrophages (AM) and neutrophils play cardinal roles in the pathogenesis of the inflammatory lung lesion. The factors responsible for the induction and perpetuation of the neutrophilic alveolitis are not known. Recently, a novel cytokine (Interleukin-8) was described that is released by activated mononuclear phagocytes and a variety of other cell types, and it exhibits potent chemotactic activity for polymorphonuclear leukocytes (PMN). Increased expression of IL-8 has been described in other inflammatory disorders characterized by neutrophilic infiltration, including psoriasis, rheumatoid arthritis, and the sepsis syndrome, but no studies have assessed this cytokine in the context of interstitial pulmonary disorders. We have previously shown that normal human AM release IL-8 upon appropriate stimulation, but data assessing the expression of IL-8 by human AM in specific pulmonary disease states are lacking. In this study, we examined the expression of steady-state mRNA for IL-8 by human alveolar macrophages obtained by bronchoalveolar lavage (BAL) from patients with idiopathic pulmonary fibrosis (IPF) or sarcoidosis and from healthy volunteers. Because it is known that adherence to plastic culture plates may up-regulate gene expression for IL-8 in the absence of additional stimulation, we extracted mRNA immediately from the cell pellet obtained by BAL rather than using cultured alveolar macrophage monolayers. Northern blot analysis was performed to determine IL-8 mRNA expression. We found that BAL cells from patients with IPF constitutively expressed mRNA for IL-8, and the amount of IL-8 mRNA (as assessed by laser densitometry) correlated with the percent of neutrophils on BAL.(ABSTRACT TRUNCATED AT 250 WORDS)

Impairment of chemotaxis of polymorphonuclear leukocytes from lead acid battery workers

Since lead impairs in vitro the functions of macrophagic cells, we have studied the chemotactic activity of polymorphonuclear leukocytes (PMNs) obtained from lead acid battery workers who were removed from exposure one month before, because they had an abnormal lead absorption. Controls were 18 age matched subjects without any history of occupational lead exposure. Both lead acid battery workers and controls had no alterations of the blood haematological and metabolic parameters. Chemotaxis was carried on in Boyden chambers using zymosan activated serum as chemotactic stimulus. The chemotactic indexes are 56.4 ± 8.7 in acid battery workers and 75.6 ± in controls. The difference, which is statistically significant, shows that lead workers have an impairment of PMNs chemotactic activity.

Effect of Bacteroides fragilis cellular components on chemotactic activity of polymorphonuclear leukocytes towards Escherichia coli

Chemotaxis of polymorphonuclear leukocytes (PMNL) in response to cell components of Bacteroides fragilis alone or in combination with Escherichia coli was evaluated. E. coli produced much more powerful chemotactic factors than B. fragilis. The culture filtrate (CF), outer membrane (OM) preparation, and lipopolysaccharide (LPS) of B. fragilis slightly stimulated chemotactic activity of PMNL. The culture filtrate and OM preparation were capable of inhibiting the chemotaxis of PMNL in response to the chemotactic factors of E. coli but LPS of B. fragilis was not able to do so. Reduction by B. fragilis of PMNL chemotaxis in response to E. coli was not specific for B. fragilis but also occurred in the presence of facultative bacteria. In parallel with chemotaxis, lysozyme release, but not beta-glucuronidase release, by PMNL was significantly stimulated by E. coli but not by B. fragilis.

Reduction of total hemolytic complement activity with Naja haje cobra venom factor does not prevent endotoxin-induced lung injury in sheep.

We studied the effects of reducing total hemolytic complement activity with Naja haje cobra venom factor on the lung injury caused by intravenously infused endotoxin in 5 unanesthetized sheep with lung lymph fistulas. In normal sheep, infusions of lipopolysaccharide W from Escherichia coli (1.0 micrograms/kg) intravenously over 30 min caused increases in protein-rich lung lymph flow as well as the appearance in plasma and lung lymph of complement (C5)-derived chemotactic activity for polymorphonuclear leukocytes. Reduction of total hemolytic complement activity by treatment with Naja haje cobra venom factor (12 to 17 U/kg intraperitoneally) did not prevent the lung injury caused by endotoxin and also did not prevent the appearance in plasma and lung lymph of chemotactic activity. We conclude that although complement appears to be activated following intravenously infused endotoxin in sheep, a completely intact complement system is not necessary for endotoxin-induced lung injury.

Demonstration of leukotriene B4 in the scale extracts of psoriasis and inflammatory pustular dermatoses. Correlation with leukocyte chemotactic activity and C5a anaphylatoxin

We carried out quantification of the levels of leukotriene B4 (LTB4), a highly potent cell membrane-derived leukocyte chemotactic factor, in scale extracts of psoriasis and related inflammatory dermatoses characterized by sterile subcorneal pustule formation by using radioimmunoassay. A small amount of LTB4 was demonstrable even in extracts from non-inflammatory stratum corneum, but larger amounts were detected in the scale extract from pustular psoriasis and in those from psoriasis vulgaris. There was a significant correlation between the LTB4 and C5a levels in scale extracts, suggesting that complement activation and generation of LTB4 are a closely related event in psoriatic lesions. However, in contrast to highly significant correlation noted between the amount of C5a and chemotactic activity for polymorphonuclear leukocytes (PMN) detectable in scale extracts, LTB4 levels correlated only marginally with the chemotactic activity.

Etretinate therapy reduces polymorphonuclear leukocyte chemotaxis—enhancing properties of psoriatic serum

Using a Boyden chamber technic, we measured the directed chemotaxis of polymorphonuclear leukocytes obtained from control subjects or psoriasis patients when the leukocytes were placed in sera obtained from control subjects or psoriasis patients. The samples from patients were obtained before therapy and after 2 and 4 weeks of etretinate administration. Compared with control sera, the sera from seven untreated psoriasis patients significantly enhanced the chemotaxis of polymorphonuclear leukocytes from control subjects toward a chemotaxin (p less than 0.05). After 4 weeks of etretinate therapy, the chemotaxis-stimulating ability of the sera from psoriasis patients was no longer significantly greater than that of the control sera. This decline in the chemotaxis-stimulating activity of our patients' sera preceded significant clearing of their psoriasis. The levels of circulating etretinate in the blood of our patients could not account for the reduction. Etretinate therapy had no apparent direct effect on the chemotactic activity of polymorphonuclear leukocytes from the psoriasis patients but may act in part by reducing the inflammatory effects of psoriatic sera.

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.

Production of chemotactic activity for polymorphonuclear leukocytes by cultured rat hepatocytes exposed to ethanol.

Acute alcoholic hepatitis is characterized by infiltration of the liver parenchyma with polymorphonuclear leukocytes. As a possible explanation for this phenomenon, we have found that ethanol stimulates cultured rat hepatocytes to generate potent chemotactic activity. Hepatocytes (greater than 99% pure), isolated from the livers of Sprague-Dawley rats, responded to incubation with ethanol (2.0-10 mM) by releasing chemotactic activity for human polymorphonuclear leukocytes into culture supernatants in a time- and concentration-dependent fashion. Chemotactic activity was maximal after incubation of hepatocytes with 10 mM ethanol for 6 h. It was undetectable in the absence of ethanol and was reduced in the presence of either the alcohol dehydrogenase inhibitor, 4-methylpyrazole, or the acetaldehyde dehydrogenase inhibitor, cyanamide. Ethanol failed to stimulate generation of chemotactic activity by either rat dermal fibroblasts, hepatic sinusoidal endothelial cells, or Kupffer cells. The chemotactic activity generated by ethanol-treated rat hepatocytes was recovered from culture supernatants in the lipid phase after extraction with chloroform/methanol. Thin-layer chromatography and high performance liquid chromatography of chloroform/methanol extracts demonstrated that the chemotactic factor probably is a polar lipid. This chemotactic lipid may account, in part, for the leukocytic infiltration of the liver parenchyma that is observed during the course of acute alcoholic hepatitis.

Characterization of the effect of influenza virus on polymorphonuclear leukocyte membrane responses

Depressed chemotactic activity of polymorphonuclear leukocytes (PMNL) infected with influenza virus could be due to changes occurring at the plasma membrane. The present study examined the effect of unopsonized influenza virus on chemotaxis, adherence, receptor binding, shape change, membrane fluidity, and release of specific granules from PMNL. Chemotactic activity of PMNL under-agarose to the chemoattractants, zymosan-activated serum ( ZAS ) and N-formyl-methionyl-leucyl- phenylalanine (fMLP), and adherence of PMNL to a plastic surface were markedly decreased in virus-treated cells as compared to control cells. The binding of fMLP to the PMNL was increased in virus-treated cells compared with control cells. Exposure of cells to virus, ZAS , or fMLP caused 35%-50% of the cells to become bipolar in shape, whereas less than 5% of the cells exposed to buffer became bipolar. Influenza virus did not alter membrane fluidity as measured by electron spin resonance spectroscopy with the probe 5-doxyl stearate. Virus-treated PMNL stimulated with FMLP or Staphylococcus aureus exhibited a marked decrease in the amount of lactoferrin released into phagosomes, onto the cells' outer membrane, and into the extracellular medium as compared to control cells. The possible relationship between inhibition of lysosomal enzyme degranulation and decreased chemotactic activity and adherence of PMNL is discussed.

Characterization of the Effect of Influenza Virus on Polymorphonuclear Leukocyte Membrane Responses

Depressed chemotactic activity of polymorphonuclear leukocytes (PMNL) infected with influenza virus could be due to changes occurring at the plasma membrane. The present study examined the effect of unopsonized influenza virus on chemotaxis, adherence, receptor binding, shape change, membrane fluidity, and release of specific granules from PMNL. Chemotactic activity of PMNL under-agarose to the chemoattractants, zymosan-activated serum ( ZAS ) and N-formyl-methionyl-leucyl-phenylalanine (fMLP), and adherence of PMNL to a plastic surface were markedly decreased in virus-treated cells as compared to control cells. The binding of fMLP to the PMNL was increased in virus-treated cells compared with control cells. Exposure of cells to virus, ZAS , or fMLP caused 35%-50% of the cells to become bipolar in shape, whereas less than 5% of the cells exposed to buffer became bipolar. Influenza virus did not alter membrane fluidity as measured by electron spin resonance spectroscopy with the probe 5-doxyl stearate. Virus-treated PMNL stimulated with FMLP or Staphylococcus aureus exhibited a marked decrease in the amount of lactoferrin released into phagosomes, onto the cells' outer membrane, and into the extracellular medium as compared to control cells. The possible relationship between inhibition of lysosomal enzyme degranulation and decreased chemotactic activity and adherence of PMNL is discussed.

Complement and endotoxin-induced lung injury in sheep.

Intravenous infusions of endotoxin in sheep cause lung injury characterized by edema due to increased microvascular permeability. Similar increases in pulmonary microvascular permeability are seen in septic patients with the adult respiratory distress syndrome. Since endotoxin-induced lung injury may be mediated by interactions between products of complement activation and polymorphonuclear leukocytes, plasma and lung lymph from six unanesthetized sheep infused with Escherichia coli endotoxin (1.0 micrograms/kg over 30 min) were examined for complement-derived chemotactic activity. By 2-3 hr following infusion of endotoxin, all animals had the increased lung lymph fluid and protein flows characteristic of permeability edema. Preinfusion samples of plasma and lung lymph did not contain chemotactic activity for polymorphonuclear leukocytes. Following infusion of endotoxin, however, significant chemotactic activity was detected in plasma at 0.5-3.5 hr (P less than 0.05) and in lymph at 1.5-6.5 hr (P less than 0.025). The chemotactic activity was heat stable (56 degrees C for 30 min) but was abolished by treatment with antibodies to C5. These data indicate that infusions of endotoxin lead to the generation in plasma, and the appearance in lung lymph, of C5-derived peptides with chemotactic activity for polymorphonuclear leukocytes. C5-derived peptides may account for the pulmonary microvascular leukostasis and endothelial injury that lead to increased permeability edema after infusions of endotoxin.