Chemotaxis Of Human Leukocytes Research Articles

Roles of Islet Toll-Like Receptors in Pig to Mouse Islet Xenotransplantation

Although innate immunity plays important roles in xenograft rejection, there have been few studies on the role of toll-like receptors (TLRs) in xenotransplantation. Furthermore, most studies focused on the recipient's TLRs. Therefore, we investigated whether TLRs in porcine islets can contribute to islet xenograft rejection. Adult porcine islets were isolated and stimulated by polyinosinic/polycytidylic acid (poly I:C) or lipopolysaccharide (LPS). Both poly I:C and LPS stimulation in porcine islets induced expression of chemokines (RANTES, MCP-1, IP-10, and IL-8), cytokines (IL-6 and type I interferons), and adhesion molecules (VCAM-1 and ICAM-1). Porcine islet supernatants stimulated by TLR agonists induced chemotaxis of human leukocytes. They also induced procoagulant activation (tissue factor and fgl-2). However, TLR stimulation did not influence insulin secretion. When porcine MyD88 was knocked down using shRNA lentivirus, TLR-mediated induction of proinflammatory mediators and procoagulants was attenuated. When LPS was injected to MyD88 or TLR4 knockout mice after porcine islet transplantation, LPS stimulation on donor islets interfered with islet xenograft tolerance induction by anti-CD154 antibodies. Inflammatory cell infiltration and expression of proinflammatory chemokines and cytokines in islet xenografts also increased. In conclusion, TLR activation in porcine islets induced both a proinflammatory and procoagulant response and thereby contributed to xenograft rejection.

Characterization of AQX‐1125, a small‐molecule SHIP1 activator

The SH2-containing inositol-5'-phosphatase 1 (SHIP1) metabolizes PI(3,4,5)P3 to PI(3,4)P2. SHIP1-deficient mice exhibit progressive inflammation. Pharmacological activation of SHIP1 is emerging as a potential therapy for pulmonary inflammatory diseases. Here we characterize the efficacy of AQX-1125, a small-molecule SHIP1 activator currently in clinical development. The effects of AQX-1125 were tested in several in vitro assays: on enzyme catalytic activity utilizing recombinant human SHIP1, on Akt phosphorylation in SHIP1-proficient and SHIP1-deficient cell lines, on cytokine release in murine splenocytes, on human leukocyte chemotaxis using modified Boyden chambers and on β-hexosaminidase release from murine mast cells. In addition, pharmacokinetic and drug distribution studies were performed in rats and dogs. AQX-1125 increased the catalytic activity of human recombinant SHIP1, an effect, which was absent after deletion of the C2 region. AQX-1125 inhibited Akt phosphorylation in SHIP1-proficient but not in SHIP1-deficient cells, reduced cytokine production in splenocytes, inhibited the activation of mast cells and inhibited human leukocyte chemotaxis. In vivo, AQX-1125 exhibited >80% oral bioavailability and >5 h terminal half-life. Consistent with the role of SHIP1 in cell activation and chemotaxis, the SHIP1 activator AQX-1125 inhibits Akt phosphorylation, inflammatory mediator production and leukocyte chemotaxis in vitro. The in vitro effects and the pharmacokinetic properties of the compound make it a suitable candidate for in vivo testing in various models of inflammation.

Imaging G-protein Coupled Receptor (GPCR)-mediated Signaling Events that Control Chemotaxis of <em>Dictyostelium Discoideum</em>

Many eukaryotic cells can detect gradients of chemical signals in their environments and migrate accordingly (1). This guided cell migration is referred as chemotaxis, which is essential for various cells to carry out their functions such as trafficking of immune cells and patterning of neuronal cells (2, 3). A large family of G-protein coupled receptors (GPCRs) detects variable small peptides, known as chemokines, to direct cell migration in vivo (4). The final goal of chemotaxis research is to understand how a GPCR machinery senses chemokine gradients and controls signaling events leading to chemotaxis. To this end, we use imaging techniques to monitor, in real time, spatiotemporal concentrations of chemoattractants, cell movement in a gradient of chemoattractant, GPCR mediated activation of heterotrimeric G-protein, and intracellular signaling events involved in chemotaxis of eukaryotic cells (5-8). The simple eukaryotic organism, Dictyostelium discoideum, displays chemotaxic behaviors that are similar to those of leukocytes, and D. discoideum is a key model system for studying eukaryotic chemotaxis. As free-living amoebae, D. discoideum cells divide in rich medium. Upon starvation, cells enter a developmental program in which they aggregate through cAMP-mediated chemotaxis to form multicullular structures. Many components involved in chemotaxis to cAMP have been identified in D. discoideum. The binding of cAMP to a GPCR (cAR1) induces dissociation of heterotrimeric G-proteins into Gγ and Gβγ subunits (7, 9, 10). Gβγ subunits activate Ras, which in turn activates PI3K, converting PIP(2;) into PIP(3;) on the cell membrane (11-13). PIP(3;) serve as binding sites for proteins with pleckstrin Homology (PH) domains, thus recruiting these proteins to the membrane (14, 15). Activation of cAR1 receptors also controls the membrane associations of PTEN, which dephosphorylates PIP(3;) to PIP(2;)(16, 17). The molecular mechanisms are evolutionarily conserved in chemokine GPCR-mediated chemotaxis of human cells such as neutrophils (18). We present following methods for studying chemotaxis of D. discoideum cells. 1. Preparation of chemotactic component cells. 2. Imaging chemotaxis of cells in a cAMP gradient. 3. Monitoring a GPCR induced activation of heterotrimeric G-protein in single live cells. 4. Imaging chemoattractant-triggered dynamic PIP(3;) responses in single live cells in real time. Our developed imaging methods can be applied to study chemotaxis of human leukocytes.

Imaging G-protein Coupled Receptor (GPCR)-mediated Signaling Events that Control Chemotaxis of <em>Dictyostelium Discoideum</em>

Many eukaryotic cells can detect gradients of chemical signals in their environments and migrate accordingly 1. This guided cell migration is referred as chemotaxis, which is essential for various cells to carry out their functions such as trafficking of immune cells and patterning of neuronal cells 2, 3. A large family of G-protein coupled receptors (GPCRs) detects variable small peptides, known as chemokines, to direct cell migration in vivo 4. The final goal of chemotaxis research is to understand how a GPCR machinery senses chemokine gradients and controls signaling events leading to chemotaxis. To this end, we use imaging techniques to monitor, in real time, spatiotemporal concentrations of chemoattractants, cell movement in a gradient of chemoattractant, GPCR mediated activation of heterotrimeric G-protein, and intracellular signaling events involved in chemotaxis of eukaryotic cells 5-8. The simple eukaryotic organism, Dictyostelium discoideum, displays chemotaxic behaviors that are similar to those of leukocytes, and D. discoideum is a key model system for studying eukaryotic chemotaxis. As free-living amoebae, D. discoideum cells divide in rich medium. Upon starvation, cells enter a developmental program in which they aggregate through cAMP-mediated chemotaxis to form multicullular structures. Many components involved in chemotaxis to cAMP have been identified in D. discoideum. The binding of cAMP to a GPCR (cAR1) induces dissociation of heterotrimeric G-proteins into Gγ and Gβγ subunits 7, 9, 10. Gβγ subunits activate Ras, which in turn activates PI3K, converting PIP2 into PIP3 on the cell membrane 11-13. PIP3 serve as binding sites for proteins with pleckstrin Homology (PH) domains, thus recruiting these proteins to the membrane 14, 15. Activation of cAR1 receptors also controls the membrane associations of PTEN, which dephosphorylates PIP3 to PIP2 16, 17. The molecular mechanisms are evolutionarily conserved in chemokine GPCR-mediated chemotaxis of human cells such as neutrophils 18. We present following methods for studying chemotaxis of D. discoideum cells. 1. Preparation of chemotactic component cells. 2. Imaging chemotaxis of cells in a cAMP gradient. 3. Monitoring a GPCR induced activation of heterotrimeric G-protein in single live cells. 4. Imaging chemoattractant-triggered dynamic PIP3 responses in single live cells in real time. Our developed imaging methods can be applied to study chemotaxis of human leukocytes.

Immunological consequences of cross‐species infection of porcine endothelial cells with human cytomegalovirus

Human cytomegalovirus (HCMV) infection or reactivationrepresents one of the most important infectious disease problems in allotransplantation. Transmission and activation of porcine cytomegalovirus in pig‐to‐baboon xenotransplantation is associated with xenograft injury and possibly an increased incidence of consumptive coagulopathy. However, stringent protocols used in the rearing of pig donors practically exclude a risk of porcine CMV. In contrast, in pig‐to‐human xenotransplantation, the porcine endothelium will inevitably be exposed to human pathogens such as HCMV, and the consequences of potential HCMV cross‐species infection of porcine xenografts, in particular endothelial injury and immune activation, remain to be elucidated. Since it is of outmost importance to anticipate any detrimental consequence of this contact, the susceptibility of porcine endothelial cells (pEC) to HCMV infection and its effect on cell surface molecule expression and human leukocyte recruitment was evaluated.Clinical and laboratory HCMV strains, including an EC propagated (TB40/E) and two fibroblast‐adapted (AD169 and TB40/F) strains, as well as UV‐inactivated, or heparin‐treated controls were inoculated to a panel of pEC stemming from different vascular beds.Infection at various MOI was analyzed by PCR, immediate early (IE), early and late antigen expression. To determine HCMV cytopathogenicity, morphological changes were monitored using live cell imaging and apoptosis was analyzed by flow cytometry. The capability of pEC to produce infectious virus was quantified by focus expansion assays. After inoculation, cell surface receptor expression was studied by flow cytometry on pEC bulk cultures and also differentially on IE‐positive (infected) and IE‐negative (bystander) pEC. Adhesion of human PBMC was tested on HCMV infected pEC monolayers and cell‐free pEC supernatants were analyzed for their cytokine content, chemotactic activity, and stimulatory effect on resting secondary pEC cultures.All viral strains infected pEC with differences in the infection efficiency (percentage of IE‐positive cells) and the kinetics of cytopathology. Moreover, differences in susceptibility of pEC derived from distinct vascular beds were observed. HCMV underwent a complete replication cycle in about 5% of the infected pEC. Comparing the permissiveness of pEC to human aortic EC (HAEC) revealed differences in strain susceptibility and lower rates of late antigen expression in pEC. Finally, HCMV‐infected pEC were demonstrated to release viral progeny with a lower efficiency than infected HAEC.Cell‐surface expression of E‐selectin and VCAM‐1 was upregulated not only in the IE‐positive fractions (infected) of HCMV‐inoculated cultures but also in the IE‐negative fractions, presumably by bystander effects. In contrast, porcine MHC class I expression was downregulated in IE‐positive, and upregulated in IE‐negative cells. The receptor alterations in the IE‐negative fraction were mediated by pEC‐derived soluble factors and consequently both porcine IL‐6 and IL‐8 were detected in the supernatants. The increased adhesion receptor expression was paralleled by enhanced human leukocyte chemotaxis and adhesion to infected pEC cultures. Pretreatment of HCMV with heparin blocking viral entry, but not UV‐inactivation blocking viral replication, prevented adhesion‐receptor modulation, and reversed the increased adhesion and chemotaxis.In summary, HCMV may productively infect pECfollowing pig‐to‐human solid organ transplantation and may alter and activate the porcine endothelium, rendering the xenograft more susceptible to human leukocyte recruitment and rejection. Therefore, understanding the mechanisms of cross‐species HCMV infection and finding strategies to render pEC resistant to HCMV infection willbe essential for xenotransplantation to become a clinical reality.

Human CMV Infection of Porcine Endothelial Cells Increases Adhesion Receptor Expression and Human Leukocyte Recruitment

Potential xenozoonosis is a concern for the clinical application of xenotransplantation. Human cytomegalovirus (HCMV) is one of the most important pathogens in allotransplantation, but the consequences of HCMV cross-species infection of porcine xenografts are unknown. Therefore, we investigated the effects of HCMV infection of porcine endothelial cells (pEC) on cell surface molecule expression and human leukocyte recruitment. Infection of pEC inoculated with untreated, UV-inactivated, or heparin-treated HCMV at a multiplicity of infection (MOI) of 1 was analyzed by immediate early (IE) antigen expression. Cell surface receptor expression was studied by flow cytometry on pEC bulk cultures and differentially on IE-positive and -negative pEC. Adhesion of human leukocytes was tested on pEC monolayers. pEC supernatants were analyzed for cytokine content, chemotactic activity, and stimulatory effect on resting secondary pEC cultures. At day 2 postinfection, IE staining was evident in 10% to 20% of HCMV-infected cells. Cell-surface expression of E-selectin and vascular cell adhesion molecule-1 (VCAM-1) was upregulated in both IE-negative and -positive fractions of HCMV-infected pEC. In contrast, porcine major histocompatibility complex class I expression was upregulated in IE-negative cells, but reduced in IE-positive cells. The receptor alterations in the IE-negative fraction were mediated by pEC-derived soluble factors. The increased adhesion receptor expression was paralleled by enhanced human leukocyte chemotaxis and adhesion to infected pEC cultures. Pretreatment of HCMV with heparin, but not UV-inactivation, prevented adhesion-receptor modulation and reversed the increased adhesion and chemotaxis. After pig-to-human solid organ transplantation HCMV may infect and activate the porcine endothelium, rendering the xenograft more susceptible to human leukocyte recruitment and rejection.

Inhibition of HIV-1 infection by viral chemokine U83A via high-affinity CCR5 interactions that block human chemokine-induced leukocyte chemotaxis and receptor internalization

AbstractHIV-1 strains use C-C-chemokine receptor 5, CCR5, as a coreceptor for host transmission. Human CCR5 chemokine ligands inhibit binding and infection, whereas CCR5 mutations also inhibit infection by preventing surface expression, resulting in delayed progression to AIDS. Here, we describe a human herpesvirus 6 (HHV-6A) chemokine, U83A, which binds CCR5 with higher affinity than human chemokines, displacing their binding and leading to inhibition of chemotaxis of human leukocytes. Similarly, U83A inhibits infection by HIV-1 strains which use CCR5, but not the CXCR4, coreceptor. Unlike human CCR5 chemokine ligands which induce rapid CCR5 internalization mediated via clathrin, treatment with U83A prevents internalization. A spliced truncated U83A isoform, U83A-N, also binds CCR5 albeit with lower affinity, and this correlates with lower HIV-1 infection inhibition, whereas further truncation abolishes binding and any inhibition. Confocal microscopy confirms CCR5 internalization inhibition by U83A treatment, whereas labeled transferrin uptake shows that endocytosis via clathrin is unaltered. Previous results show that, although U83A-N is an antagonist, U83A is an agonist for CCR1, CCR4, CCR6, and CCR8 present on immune effector and antigen-presenting cells and here also shown for CCR5. Thus, U83A could act as a novel inhibitor of HIV-1 infection while also stimulating local immunity to the virus.

In Vitro Antichemotactic Activity of Chamomilla recutita Hydroethanol Extract

In this study, we have investigated the ability of the hydroethanolic extract prepared from the flower heads of Chamomilla recutita. (L.) Rauschert (Asteraceae) to interfere with human leukocyte chemotaxis induced by casein using the in vitro. Boyden system. The results showed a striking dose-related inhibition of the casein-induced human polymorphonuclear leukocyte migration after chamomile extract treatment in comparison with untreated controls, a biological effect never reported before for this ancient plant. Moreover, these effects were similar to those showed by dexamethasone, a model drug. Of particular interest are the results observed after pretreating these cells with morphine, in which the antichemotactic activity could be partially blocked, suggesting that these effects might be opioid receptor mediated. The data herein presented may be useful to clarify the worldwide use of this herbal medicine as an oral infusion, supporting its ethnomedical application as a calming, spasmolytic, and anti-inflammatory agent.

Comparison of chemical constituents of Chamomilla recutita (L.) rauschert essential oil and its anti-chemotactic activity

Several essential oil samples from dried flower-heads of Chamomilla recutita (L.) Rauschert, grown and commercially available around Curitiba metropolitan area, South of Brazil, were analyzed by GC-MS and the chemical constituents were compared with an Egyptian sample obtained under similar conditions and used as a control. The local grown herbs showed levels of essential oil below the standards recommended by the Brazilian Pharmacopoeia. Also, differences in their composition as well as in the quantity of several components were found such as the unexpected inversion of the relative constitution of the A and B alpha-bisabolol oxides. Of particular interest was the striking effect of the chamomile extracts upon human leukocyte chemotaxis, a biological anti-inflammatory activity not reported before, in which cell migration was in vitro inhibited at the same level as showed by dexamethasone.

Brugia malayiAsparaginyl–Transfer RNA Synthetase Induces Chemotaxis of Human Leukocytes and Activates G‐Protein–Coupled Receptors CXCR1 and CXCR2

Background. Lymphatic filariasis is a chronic human parasitic disease in which the parasites repeatedly provoke acute and chronic inflammatory reactions in the host bloodstream and lymphatics. Excretory-secretory products derived from filariae are believed to play an important role in the development of associated immunologic conditions; however, the specific mechanisms involved in these changes are not well understood. Recently, human cytoplasmic aminoacyl-transfer (t) RNA synthetases, which are autoantigens in idiopathic inflammatory myopathies, were shown to activate chemokine receptors on T lymphocytes, monocytes, and immature dendritic cells by recruiting immune cells that could induce innate and adaptive immune responses. Filarial (Brugia malayi) asparaginyl-tRNA synthetase (AsnRS) is known to be an immunodominant antigen that induces strong human immunoglobulin G3 responses.Methods. Recombinant B. malayi AsnRS was used to perform cellular function assays--for example, chemotaxis and kinase activation assays.Results. Unlike human AsnRS, parasite AsnRS is chemotactic for neutrophils and eosinophils. Recombinant B. malayi AsnRS but not recombinant human AsnRS induced chemotaxis of CXCR1 and CXCR2 single-receptor-transfected HEK-293 cell lines, blocked CXCL1-induced calcium flux, and induced mitogen-activated protein kinase.Conclusions. Our findings suggest that a filarial parasite chemoattractant protein may contribute to the development of chronic inflammatory disease and that chemokine receptors may be therapeutic targets to ameliorate parasite-induced pathology.

Parthenolide and its photochemically synthesized 1(10)Z isomer: chemical reactivity and structure–activity relationship studies in human leucocyte chemotaxis

The present study has achieved the photochemical conversion of a germacrolide into a melampolide. The investigation on their chemical properties allowed us to evaluate the minimum interatomic distance needed for transannular bridging of C10 ring in germacrolides and to explain the regiochemical selectivity of electrophilic cyclizations. The antiinflammatory activity of parthenolide and its semisynthetic derivatives was evaluated by in vitro chemotaxis assay with human neutrophiles. These structure–activity relationship studies have led to hypothesize a new pharmacophore and have provided useful information for computationally designed drugs.

Influence of acyl and plasmalogenic analogs of platelet activating factor on chemotaxis of human leukocytes in vitro and their inflammatory and antiinflammatory activity in vivo.

The effects of platelet activating factor (PAF) and its cell analogs 1-O-alk-1;-enyl-2-acetyl-sn-glycero-3-phosphocholine (1-alkenyl-PAF) and 1-acyl-2-acetyl-sn-glycero-3-phosphocholine (1-acyl-PAF) on chemotaxis of human leukocytes in vitro and their inflammatory and antiinflammatory activities in vivo were studied. Both analogs stimulated chemotaxis of human leukocytes in agarose gel. PAF and 1-alkenyl-PAF induced rat paw edema in the range of doses 0.1-10 and 10-100 micro g per paw, respectively. Paw edema induced by 1-acyl-PAF (10-100 micro g per paw) was more pronounced than that induced by PAF or 1-alkenyl-PAF. The latter also exhibited significant antiinflammatory effect by inhibiting PAF- or carrageenan-induced rat paw edema, and this effect exceeded that of dexamethasone. In these models of inflammation 1-acyl-PAF did not exhibit any antiinflammatory activity. The data suggest that PAF is not the only cell phospholipid mediating inflammation--its cell analogs, 1-acyl-PAF and 1-alkenyl-PAF, may also be involved into the inflammatory response. Possible interrelationships between cellular synthesis of 1-acyl-PAF, its formation in oxidized LDL, biological effects of lysolecithin, and penetration of LDL into the arterial wall are discussed.

Characterization of chenodeoxycholic acid as an endogenous antagonist of the G-coupled formyl peptide receptors.

To demonstrate the role of bile acids in immune modulation we examined the ability of select bile acids to inhibit leukocyte migration and chemoattractant receptor function. To elucidate this mechanism, we employed primary human monocytes, neutrophils and cell lines transfected to express either the high affinity fMLP receptor (FPR) or the low affinity fMLP receptor like 1 (FPRL1). Cells were treated with chenodeoxycholic acid (CDCA) and related bile acids in a 0-400 micromolar range. Cell viability, chemotaxis and calcium flux analysis were preformed. We observed that pathophysiological levels (< or = 150 micromolar) of CDCA competitively inhibited 3H-fMLP binding to human monocytes, FPR and FPRL1 transfected cells. Additionally, CDCA reduced both the chemotactic and calcium flux responses induced by fMLP or "W" peptide. Further, CDCA inhibited anti-FPR antibody binding to monocytes. CDCA selectively inhibited human leukocyte chemotaxis and calcium flux induced by fMLP, but not other chemoattractants, suggesting a mechanism for inhibition of inflammation and suppression of innate immune response.

Phospholipase A2 is involved in chemotaxis of human leukocytes.

Recently, it was shown that phospholipase A2 (PLA2) is essential for exposing Mac-1 (CDllb/CD18) on the cell surface of leukocytes1,2. Mac-1 is mandatory for leukocyte migration in vitro, and lack of Mac-1 on human leukocytes is associated with failure of in vitro chemotaxis3. To further substantiate the contribution of PLA2 in cell migration we investigated the effects of PLA2 inhibition on chemotaxis of mixed human leukocytes (MHL) induced by fMLP and the G-protein activator NaF7–9. Migration was measured in a modified 48-well micro-chemotaxis Boyden-chamber6. As PLA2 inhibitors manoalide4, mepacrine5, and BM 16.2115, a new PLA2 inhibitor, were employed.

Anaphylactic and chemotactic response of mammalian cells to zymosan-activated shark serum

Human anaphylatoxins, C3a and C5a, are small proteins which are generated by proteolytic cleavage of the complement components C3 and C5 during activation of complement. Complement activity via the classical and alternative pathways has been described for the nurse shark (Ginglymostoma cirratum) and studies have demonstrated chemotactic activity of shark leucocytes in response to activated shark serum. However, anaphylatoxins have not been reported in an elasmobranch. This investigation was undertaken to examine activated shark serum for anaphylatoxins by studying the anaphylactic and chemotactic response of mammalian cells. Shark (G. cirratum) serum, activated with zymosan (10mgml−1) for 3h at 30°C, was fractionated on Sephadex G-100 and G-25 columns, and fractions were pooled and assayed for spasmogenic and chemotactic activity.In vitrochemotaxis assays were set up in blind well chemotaxis chambers using human leucocytes as responding cells. A significant (P<0·05) migratory response was obtained with several serum pools. Spasmogenic activity was measured by smooth muscle contraction of rat ileum, and was present in serum fractions containing low molecular size (<17·5kDa) proteins/peptides, which also induced chemotaxis of human leucocytes. No spasmogenic or chemotactic activity was demonstrated in fractions of normal (non-activated) shark serum. This study shows that mammalian cells respond to factors generated in activated shark serum and results suggest that the chemotactic and anaphylactic activity in shark serum might be due to a biologically active peptide(s) derived from the cleavage of complement component(s), perhaps analogous to mammalian C3a and/or C5a.

Stimulation of neutrophil leukocyte chemotaxis by a cloned cytolytic enterotoxin ofAeromonas hydrophila

A cytolytic enterotoxin produced by Aeromonas hydrophila, isolate SSU, has been cloned in our laboratory. This enterotoxin lysed rabbit red blood cells, destroyed Chinese hamster ovary cells, caused fluid secretion in rat ligated ileal loops, inhibited the phagocytic function of mouse phagocytes, and was lethal to mice when injected intravenously. In this study, the effect of this cytolytic enterotoxin on the chemotaxis of human leukocytes (cell line RPMI 1788) was examined. This toxin, at concentrations from 92.5 to 370 ng/ml, significantly stimulated the chemotactic activity of human leukocytes in a dose-dependent fashion. The stimulation of leukocyte chemotaxis elicited by cytolytic enterotoxin was abolished when the toxin was neutralized, heated, or exposed to low pH values. This stimulatory effect also was inhibited by various concentrations of pertussis toxin. These results demonstrated that cytolytic enterotoxin may stimulate increased chemotaxis of human leukocytes, and suggest that human leukocytes may possess cytolytic enterotoxin receptors which may be coupled to pertussis toxin-sensitive G-protein.

Stimulation of neutrophil leukocyte chemotaxis by a cloned cytolytic enterotoxin of Aeromonas hydrophila.

A cytolytic enterotoxin produced by Aeromonas hydrophila, isolate SSU, has been cloned in our laboratory. This enterotoxin lysed rabbit red blood cells, destroyed Chinese hamster ovary cells, caused fluid secretion in rat ligated ileal loops, inhibited the phagocytic function of mouse phagocytes, and was lethal to mice when injected intravenously. In this study, the effect of this cytolytic enterotoxin on the chemotaxis of human leukocytes (cell line RPMI 1788) was examined. This toxin, at concentrations from 92.5 to 370 ng/ml, significantly stimulated the chemotactic activity of human leukocytes in a dose-dependent fashion. The stimulation of leukocyte chemotaxis elicited by cytolytic enterotoxin was abolished when the toxin was neutralized, heated, or exposed to low pH values. This stimulatory effect also was inhibited by various concentrations of pertussis toxin. These results demonstrated that cytolytic enterotoxin may stimulate increased chemotaxis of human leukocytes, and suggest that human leukocytes may possess cytolytic enterotoxin receptors which may be coupled to pertussis toxin-sensitive G-protein.

Effects of two erythromycins, doxycycline and phenoxymethylpenicillin on human leucocyte chemotaxis in vitro.

The effect of erythromycin, doxycycline and phenoxymethylpenicillin on human polymorphonuclear leucocyte (PMNL) chemotaxis was studied in vitro with Boyden's filter technique. Both erythromycin base and 2'-acetyl erythromycin stearate increased the PMNL chemotaxis significantly at concentrations greater than or equal to 50 microM, 2'-acetyl erythromycin stearate also at concentrations of 2.5-5 microM. Phenoxymethyl-penicillin reduced migration at 500 microM but lower concentrations had no significant effect. Doxycycline prevented migration significantly at concentrations greater than or equal to 50 microM.

Leucocyte chemotaxis to mycetoma agents — the effect of the antifungal drugs griseofulvin and ketoconazole

Polymorphonuclear leucocytes are the predominant cell type in the inflammatory infiltrate around mycetoma grains. Human leucocyte chemotaxis using cells from healthy subjects has been demonstrated using cytoplasmic antigens from 3 mycetoma agents: Madurella mycetomatis and Pseudallescheria boydii, both fungi, and Streptomyces somaliensis, an actinomycete. There was a statistically insignificant increase in chemotaxis in the presence of ketoconazole, but griseofulvin was a potent inhibitor of leucocyte movement. Griseofulvin has been used to improve the clinical appearances in eumycetoma (fungal mycetoma) cases which respond poorly to antifungal chemotherapy and it is suggested that its effect in this condition depends, in part, on its ability to inhibit leucocyte aggregation around mycetoma grains.

Migration of human leukocytes from soft agarose droplet: a simplified method for studying chemotaxis and spontaneous migration.

Studies of in vitro chemotaxis and spontaneous migration of human leukocytes using the accepted method with the Boyden-chamber-filter are troublesome, because of the need for specially constructed vessels as well as the difficulties caused by the use of membrane filters. We describe a new and simplified method for measuring human leucocyte chemotaxis, which is a modification of the recently described underagarose migration method and which is based upon spontaneous migration of cells from a soft agarose droplet and in response to a chemotactic gradient. We examined suspensions of leukocytes, purified granulocytes, and mononuclear cells from 10 healthy normal adults and from 10 samples of cord blood using E Coli O111B4 endotoxin-activated human serum as attractant. Our results showed that the mean chemotactic indices (C.I.-chemotaxis/migration) for purified granulocytes and for mononuclear cells from normal individuals were 3.0 +/- 1.2 and 2.7 +/- 1.5, respectively. Chemotaxis was significantly reduced when unwashed leukocytes were studied, indicating a detrimental effect of autologous plasma on leukocytic response to a chemotactic stimulus in this system. Cord blood cells showed normal spontaneous migration, but significantly decreased chemotaxis. This preliminary report shows that the technique is simple, rapid, and reproducible, and can detect abnormalities of chemotaxis in both granulocytes and mononuclear cells.