N-formyl Methionyl Leucyl Phenylalanine Research Articles

Effect of Ylang-Ylang (Cananga odorata Hook. F. & Thomson) Essential Oil on Acute Inflammatory Response In Vitro and In Vivo.

The aim of this study is to evaluate the phytochemical profile, oral acute toxicity, and the effect of ylang-ylang (Cananga odorata Hook. F. & Thomson) essential oil (YEO) on acute inflammation. YEO was analyzed by gas chromatography/mass spectrometry. For in vitro tests, YEO was assessed using cytotoxicity, neutrophil chemotaxis induced by N-formyl methionyl leucyl phenylalanine (fMLP), and phagocytic activity tests. YEO was orally administered in zymosan-induced peritonitis, carrageenan-induced leukocyte rolling, and adhesion events in the in situ microcirculation model and in carrageenan-induced paw edema models. YEO (2000 mg/kg) was also tested using an acute toxicity test in Swiss mice. YEO showed a predominance of benzyl acetate, linalool, benzyl benzoate, and methyl benzoate. YEO did not present in vitro cytotoxicity. YEO reduced the in vitro neutrophil chemotaxis induced by fMLP and reduced the phagocytic activity. The oral treatment with YEO reduced the leukocyte recruitment and nitric oxide production in the zymosan-induced peritonitis model, reduced rolling and adherent leukocyte number induced by carrageenan in the in situ microcirculation model, and reduced carrageenan-induced edema and mechanical hyperalgesia. YEO did not present signs of toxicity in the acute toxicity test. In conclusion, YEO affected the leukocyte activation, and presented antiedematogenic, anti-hyperalgesic, and anti-inflammatory properties.

Analgesic and anti-inflammatory articular effects of essential oil and camphor isolated from Ocimum kilimandscharicum Gürke leaves

Ethnopharmacological relevanceLeaves from Ocimum kilimandscharicum Gürke (Lamiaceae) are popularly used against articular pain. Aim of studyThe aim of this study was to test the anti-inflammatory and anti-hyperalgesic (analgesic) properties of the essential oil and camphor isolated from O. Kilimandscharicum leaves (EOOK) in 4 models including zymosan induced-articular inflammation model in mice. Material and methodsFor in vivo models, EOOK was tested in carrageenan-induced paw edema model with oral doses of 30, 100, and 300 mg/kg (oral administration = p.o.) and in zymosan-induced articular inflammation (including knee edema, leukocyte infiltration, mechanical hyperalgesia and nitric oxide), EOOK (100 mg/kg, p. o.) and camphor (30 mg/kg, p. o.) were tested. EOOK (100 mg/kg, p. o.) was tested in the rolling and also in the adhesion of leukocytes to the mesenteric microcirculation in situ model of carrageenan induced inflammation and EOOK (1, 3, 10, 30, and 60 μg/mL) was tested in vitro against neutrophils chemotaxis induced by N-formyl methionyl leucyl phenylalanine (fMLP). ResultsThe treatment with EOOK significantly inhibited the carrageenan-induced edema, mechanical and cold hyperalgesia. Both, EOOK and camphor inhibited all articular parameters induced by zymosan. In situ intravitral microscopy analysis, EOOK significantly inhibited carrageenan-induced leukocyte rolling and adhesion. In vitro neutrophils chemotaxis, EOOK inhibited the leukocyte chemotaxis induced by fMLP. ConclusionsThe present study showed that EOOK inhibited pain and inflammatory parameters contributing, at least in part, to explain the popular use of this plant as analgesic natural agent. This study also demonstrates that camphor and some known anti-inflammatory compounds present in EOOK could contribute for analgesic and anti-inflammatory articular properties.

Chamomile decoction extract inhibits human neutrophils ROS production and attenuates alcohol-induced haematological parameters changes and erythrocytes oxidative stress in rat.

BackgroundThe aim of this study was to evaluate the protective effects of subacute pre-treatment with chamomile (Matricaria recutita L.) decoction extract (CDE) against stimulated neutrophils ROS production as well as ethanol (EtOH)-induced haematological changes and erythrocytes oxidative stress in rat.MethodsNeutrophils were isolated and ROS generation was measured by luminol-amplified chemiluminescence. Superoxide anion generation was detected by the cytochrome c reduction assay. Adult male wistar rats were used and divided into six groups of ten each: control, EtOH, EtOH + various doses of CDE (25, 50, and 100 mg/kg, b.w.), and EtOH+ ascorbic acid (AA). Animals were pre-treated with CDE extract during 10 days.ResultsWe found that CDE inhibited (P ≤ 0.0003) luminol-amplified chemiluminescence of resting neutrophils and N-formyl methionylleucyl-phenylalanine (fMLF) or phorbolmyristate acetate (PMA) stimulated neutrophils, in a dose-dependent manner. CDE had no effect on superoxide anion, but it inhibited (P ≤ 0.0004) H2O2 production in cell free system. In vivo, CDE counteracted (P ≤ 0.0034) the effect of single EtOH administration which induced (P < 0.0001) an increase of white blood cells (WBC) and platelets (PLT) counts. Our results also demonstrated that alcohol administration significantly (P < 0.0001) induced erythrocytes lipoperoxidation increase and depletion of sulfhydryl groups (−SH) content as well as antioxidant enzyme activities as superoxide dismutase (SOD), catalase (CAT), and glutathione peroxidase (GPx). More importantly, we found that acute alcohol administration increased (P < 0.0001) erythrocytes and plasma hydrogen peroxide (H2O2), free iron, and calcium levels while the CDE pre-treatment reversed increased (P ≤ 0.0051) all these intracellular disturbances.ConclusionsThese findings suggest that CDE inhibits neutrophil ROS production and protects against EtOH-induced haematologiacal parameters changes and erythrocytes oxidative stress. The haematoprotection offered by chamomile might involve in part its antioxidant properties as well as its opposite effect on some intracellular mediators such as H2O2, free iron, and calcium.

Effect of Camphor on the Behavior of Leukocytes &lt;i&gt;In vitro&lt;/i&gt; and &lt;i&gt;In vivo&lt;/i&gt; in Acute Inflammatory Response

Purpose: To evaluate the effect of camphor on acute inflammatory response by leukocytes on chemotaxis, antiedematogenic and phagocytic activities. Methods: The effect of camphor in acute inflammatory response evaluated by neutrophil chemotaxis, ear edema induced by croton oil, activity of the enzyme myeloperoxidase (MPO) and phagocytic activity of macrophages in Swiss mice. Results: Camphor treatment did not show any cytotoxicity. Camphor at 3, 10, and 30 µg/ml doses exhibited significant (p < 0.01) reduction on leukocyte migration toward N-formyl methionyl leucyl phenylalanine fMLP. Topical treatment with camphor did not reduce significant ear edema or MPO activity at any of the doses tested. However, in contrast, oral treatment with 100 and 200 mg/kg camphor significantly (p < 0.01) reduced ear edema and myeloperoxidase (MPO) activity. Additionally, the phagocytic activity of macrophages was not affected by camphor. Conclusions: These results indicate that the anti-inflammatory activity of camphor may be related to the inhibition of leukocyte migration and antiedematogenic activity.

Multireaction monitoring of 12 peptides for lowered immunity screening

A multireaction monitoring method using high-performance liquid chromatography-tandem mass spectrometry was developed for 12 target peptides for determination of endogenous peptide concentrations in human serum. Chromatographic separation conditions were optimized and recoveries for liquid-liquid extraction, solid-phase extraction (SPE), and ultrafiltration of endogenous peptides from human serum were compared, and the SPE method was selected for 12 targeted peptide extractions. The optimized SPE method gave recoveries higher than 60 % for all targeted peptides. The limit of detection was 10 ng/ml for most peptides, except for N-formyl-methionyl-leucyl-phenylalanine (NFMLP) and adrenocorticotropic hormone (ACTH) (18-39). The limit of detection for these two peptides was 1 ng/ml. The real serum samples of 25 elderly and 23 young people were analyzed using the optimized extraction and analysis method. Half of the 12 peptides were below the limit of quantification, and B-type natriuretic peptide, cholecystokinin, ACTH(7-38), substance P, NFMLP, and valyl-glutamyl-prolyl-isoleucyl-prolyl-tyrosine were quantified in the concentration range from 0.1 to 50 ng/ml. The concentration of ACTH(7-38) was significantly higher in elderly people and that of NFMLP was significantly lower in elderly people compared with young people (p < 0.0001). This result implies that there be a possible relationships between ACTH, NFMLP and lowered immunity.

Comparative evaluation of effects of chlorhexidine and tetracycline on neutrophil viability and functions in vitro

Background:Chlorhexidine (CHX) and Tetracycline (TET) are the two antimicrobial agents used in the management of periodontal infections, due to their antimicrobial potency and substantivity. The benefits and limitations of an antimicrobial agent can only be assessed by determining their relative toxicity to microbes and host cells.Objectives:(1) To detect the effects of CHX and TET on neutrophil viability and functions in vitro. (2) To compare the effects of CHX and TET on crevicular blood neutrophils.Materials and Methods:Crevicular blood was collected using 5-μl pipette, stored in EDTA-containing vacutainers and sent for evaluation of Cell Viability- Neutrophils would be mixed with 0.25 volume of 0.4% trypan blue in Hank's balanced salt solution (HBSS) and counted to assess the viability. Chemotaxis- Evaluated under agarose slides, a total of 100 μl crevicular blood Neutrophils-0.2% BSA or HBSS-0.2% containing concentration of CHX and TET. Oxidative Metabolism- would be assessed using nitro blue tetrazolium. In all experiments, three concentrations of TET (0.1%, 0.05%, 0.025%) and CHX (0.01%, 0.005%, 0.0025%) respectively were used. N-Formyl methionyl leucyl phenylalanine (1 μm) in the presence of 2 μg/ml cytochalasin B will be used as the activating agent. Neutrophils would be treated with CHX and TET similarly to that for chemotaxis.Results:TET comparatively has less deleterious effects on neutrophil functions as compared with that of CHX with statistically significant results for all parameters tested.Conclusion:From this study, it is inferred that CHX and TET do cause certain changes in neutrophil functions.

INHIBITION OF CYTOCHALASIN-PRIMED NEUTROPHILS BY HYPEROSMOLARITY

Experimental and clinical investigations using hyperosmotic solutions for resuscitation of hemorrhagic shock demonstrated modulation of the inflammatory response. Decreased postinjury hyperinflammation has been attributed to a reduction in neutrophil-mediated tissue damage. This study shows that cytoskeletal disruption with cytochalasinB did not reverse or prevent the inhibitory effect of an osmolarity increase on the neutrophil cytotoxic response to a formyl peptide. In cytochalasin-primed neutrophils, the hyperosmolarity-dependent inhibition promptly reversed after returning to iso-osmotic levels. Paradoxically, an increase in osmolarity after stimulation produced an increase in the release of reactive oxygen species to the extracellular medium. The inhibitory effect of hyperosmotic NaCl can be reproduced by solutions of similar osmolarity containing N-methyl glucamine or sucrose, but solutions containing mannitol allowed an almost complete response to N-formyl methionyl leucyl phenylalanine. The effects on the release of reactive oxygen species to the extracellular media found with the OxyBURST-bovine serum albumin assay correlated with the changes of the intracellular calcium signal, indicating that the inhibition by hyperosmolarity occurs near the receptor level.

Effect of normal and preeclamptic plasma on superoxide-anion production of neutrophils from healthy non-pregnant women

This study has examined whether production of superoxide-anion by granulocytes differs between non-pregnant, healthy pregnant and preeclamptic pregnant women. First, we assessed superoxide-anion production in 13 non-pregnant women, 11 healthy pregnant women and 14 preeclamptic pregnant women. Then, we examined the effect of plasma samples of healthy pregnant and preeclamptic pregnant women on superoxide production by neutrophils separated from healthy pregnant women. Superoxide generation was measured by ferricytochrome- c reduction. Phorbol-12,13-dibutyrate- and n-formyl–methionyl–leucyl–phenylalanine-stimulated superoxide-anion production was significantly decreased in healthy pregnant women's granulocytes compared with non-pregnant women. There was no significant difference between granulocyte superoxide-anion production in preeclamptic pregnant and non-pregnant women. When neutrophils from non-pregnant women were incubated in plasma from healthy pregnant women, the granulocyte phorbol-12,13-dibutyrate-stimulated superoxide-anion production was significantly inhibited. With the same stimulator, there were no significant differences between superoxide-anion production of neutrophils incubated in autologous, non-pregnant and preeclamptic pregnant plasma. If n-formyl–methionyl–leucyl–phenylalanine was used for stimulation, there were no significant differences in the superoxide-anion production of granulocytes in either group. Granulocyte superoxide-anion production decreases during pregnancy; this decrease does not occur in preeclampsia, and may cause endothelial damage. It is conceivable that there are unidentified factors in maternal circulation which inhibit superoxide-anion production by granulocytes in healthy pregnant women.

Role of osteopontin in neutrophil function

Osteopontin (OPN) is important for the function of fibroblasts, macrophages and lymphocytes during inflammation and wound healing. In recent studies of experimental colitis we demonstrated exacerbated tissue destruction in OPN-null mice, associated with reduced tumour necrosis factor-alpha expression and increased myeloperoxidase activity. The objective of this investigation therefore was to determine the importance of OPN expression in neutrophil function. Although, in contrast to macrophages, neutrophils expressed low levels of OPN with little or no association with the CD44 receptor, intraperitoneal recruitment of neutrophils in OPN-null mice was impaired in response to sodium periodate. The importance of exogenous OPN for neutrophil recruitment was demonstrated by a robust increase in peritoneal infiltration of PMNs in response to injections of native or recombinant OPN. In vitro, OPN(-/-) neutrophils exhibited reduced chemokinesis and chemotaxis towards N-formyl methionyl leucyl phenylalanine (fMLP), reflecting a reduction in migration speed and polarization. Exogenous OPN, which was chemotactic for the neutrophils, rescued the defects in polarization and migration speed of the OPN(-/-) neutrophils. In contrast, the defensive and cytocidal activities of OPN(-/-) neutrophils, measured by assays for phagocytosis, generation of reactive oxygen species, cytokine production and matrix metalloproteinase-9, were not impaired. These studies demonstrate that, while exogenous OPN may be important for the recruitment and migration of neutrophils, expression of OPN by neutrophils is not required for their destructive capabilities.

Identification of an alternative Gαq-dependent chemokine receptor signal transduction pathway in dendritic cells and granulocytes

CD38 controls the chemotaxis of leukocytes to some, but not all, chemokines, suggesting that chemokine receptor signaling in leukocytes is more diverse than previously appreciated. To determine the basis for this signaling heterogeneity, we examined the chemokine receptors that signal in a CD38-dependent manner and identified a novel “alternative” chemokine receptor signaling pathway. Similar to the “classical” signaling pathway, the alternative chemokine receptor pathway is activated by Gαi2-containing Gi proteins. However, unlike the classical pathway, the alternative pathway is also dependent on the Gq class of G proteins. We show that Gαq-deficient neutrophils and dendritic cells (DCs) make defective calcium and chemotactic responses upon stimulation with N-formyl methionyl leucyl phenylalanine and CC chemokine ligand (CCL) 3 (neutrophils), or upon stimulation with CCL2, CCL19, CCL21, and CXC chemokine ligand (CXCL) 12 (DCs). In contrast, Gαq-deficient T cell responses to CXCL12 and CCL19 remain intact. Thus, the alternative chemokine receptor pathway controls the migration of only a subset of cells. Regardless, the novel alternative chemokine receptor signaling pathway appears to be critically important for the initiation of inflammatory responses, as Gαq is required for the migration of DCs from the skin to draining lymph nodes after fluorescein isothiocyanate sensitization and the emigration of monocytes from the bone marrow into inflamed skin after contact sensitization.

Anti-formyl peptide antibodies

Antibodies that selectively bind to N-formylmethionyl leucyl phenylalanine (fMLF, also known as fMLP) have been generated. These antibodies bound to fMLF with higher affinity than to non-formylated peptide MLF: the differences in the binding energies between fMLF and MLF were 1.4–>2.1 kcal/mol.

Functional Expression ofN-Formyl Peptide Receptors in Human Bone Marrow-Derived Mesenchymal Stem Cells

Tissue injury enhances homing and engraftment of mesenchymal stem cells (MSCs). However, the mechanisms by which MSCs sense the signals released by injured tissues and migrate toward injury sites have not been fully defined. In the current report, we investigated whether human MSCs express the N-formyl peptide receptor (FPR) and the formyl peptide receptor-like-1 (FPRL1). These receptors bind to N-formylated peptides by which phagocytes migrate to inflammatory sites and fibroblasts repopulate wounds to remodel the damaged tissues. Reverse-transcription polymerase chain reaction (PCR) demonstrated that MSCs express both FPR and FPRL1 at the transcriptional level. Flow cytometric analyses revealed expression of both receptors at the protein level. Fusion of the enhanced green fluorescence protein (eGFP) to the C terminus of each receptor showed localization to the cell surface. Moreover, MSCs responded to stimulation by N-formyl methionyl leucyl phenylalanine (fMLP), a prototypic N-formyl peptide, demonstrating rapid intracellular calcium mobilization that can be blocked by pertussis toxin or cyclosporin H. It is noteworthy that the fMLP-stimulated MSCs had an enhanced adhesion to extracellular matrix protein-coated surfaces. In addition, MSCs migrated toward gradients of increasing fMLP concentration, indicating that the receptors were functionally involved in positive chemotaxis to formylated peptides. Therefore, the N-formyl peptide receptors present in MSCs may play an important role in signaling stem cell adhesion, migration, and homing to injured and inflamed tissue for repair. Such a mechanism could potentially be exploited to direct the stem cells to target specific tissue sites, such as cystic fibrosis lungs, for therapy. Disclosure of potential conflicts of interest is found at the end of this article.

Major histocompatibility complex class II (DR) antigen and costimulatory molecules on in vitro and in vivo activated human polymorphonuclear neutrophils

We have previously shown that normal human peripheral blood polymorphonuclear neutrophils (PMNs) contain cytoplasmic 'stores' of three key molecules normally associated with antigen presentation and T-cell costimulation, i.e. major histocompatibility complex class II (DR) antigen, CD80 (B7-1) and CD86 (B7-2). These cytoplasmic molecules were found to translocate to the cell surface within a few minutes following cross-linking (X-L) of Mac-1: an early neutrophil activation signal. In this study we have compared X-L of Mac -1 in parallel with four other well documented in vitro neutrophil activators: phorbol myristate acetate, N-formyl methionyl leucyl phenylalanine, lipopolysaccharide, and phagocytosis of immunoglobulin G-Latex particles. In addition, we have used paired samples of neutrophils obtained from peripheral blood (as a control) and synovial fluid from patients with rheumatoid arthritis as a source of in vivo activated cells. With the exception of phagocytosis, all activators resulted in the rapid (within 30 min) generation of two populations of activated neutrophils (designated P1 and P2) based on flow-cytometry measurements of size, granularity and phenotype. Significant up-regulation of DR and costimulatory molecules was observed, predominantly on P2 cells, with all activators except phagocytosis. CD80 and CD86 were noted to respond to the various activation signals in a different pattern suggesting that their intracellular granule location may be different. Dual-staining confocal laser microscopy studies showed that CD80 is largely confined to secretory vesicles (SVs) while CD86 appears to have a much wider distribution being found in SVs and within secondary (specific) and primary (azurophilic) granules. Increased surface expression of these antigens was also observed on P2 synovial fluid neutrophils appearing as large heterogeneous clusters on the cell surface when visualized by confocal laser microscopy.

Do Native and Polymeric α1-Antitrypsin Activate Human Neutrophils In Vitro?

alpha(1)-Antitrypsin (AAT)-Z deficiency is a risk factor for the development of COPD. Compared to wild-type M, AAT-Z has an increased tendency to polymerize, rendering it inactive as a serine proteinase inhibitor. It has been demonstrated that wild-type M- and Z-deficiency AAT polymers are chemotactic for human neutrophils. However, our own studies dispute a proinflammatory role for polymerized AAT-M and AAT-Z, suggesting rather that they are predominantly antiinflammatory, exhibiting inhibitory effects on lipopolysaccharide-stimulated human monocyte activation. The discrepancies between these observations prompted us to re-examine the effects of AAT. The effects of native and polymerized AAT-M and AAT-Z with varying levels of endotoxin contamination (0.08 to 2.55 endotoxin units [EU]/mg protein) on human neutrophil chemotaxis and interleukin (IL)-8 release, in vitro, were evaluated. Neither native nor polymerized (M- or Z-deficient) AAT contaminated with low levels of endotoxin (</= 0.08 EU/mg protein) stimulated neutrophil chemotaxis, whereas N-formyl methionyl leucyl phenylalanine (fMLP), a positive control, increased chemotaxis fourfold. A small but nonsignificant increase in neutrophil chemotaxis, however, was observed with AAT preparations containing higher levels of endotoxin (>/= 0.88 EU/mg protein), and significant chemotaxis occurred when AAT was spiked with either endotoxin or zymosan. In support, native and polymeric AAT-M with low endotoxin contamination completely inhibited neutrophil IL-8 release triggered by the zymosan, while AATs with high endotoxin contamination strongly induced IL-8 release and did not inhibit zymosan-stimulated IL-8 release. The proinflammatory effects of native and polymeric AAT may be critically dependent on the presence of other cell activators, bacterial or otherwise, while pure preparations of AAT appear to exert predominantly antiinflammatory activity.

347. Expression of the N-Formyl Peptide Receptor in Bone Marrow-Derived Human Mesenchymal Stem Cells

Human mesenchymal stem cells (hMSCs) from adult bone marrow can differentiate into a variety of different cell types including airway epithelial cells, suggesting the theraputic potential of stem cells in tissue injury repair. However, approaches for the efficient recruitmentof hMSCs to specific sites have not been established. To this end, we have investigated whether hMSCs express the N-formyl peptide receptor (FPR), which is typically expressed in polymorphonuclear leukocytes and is responsible for the neutrophil chemotactic response. Reverse transcription PCR using FPR specific primers resulted in a positive amplification of this mRNA. Sequencing of the amplicon proved 100% homology to the published FPR sequence. Immunofluorescence staining using a monoclonal antibody against human FPR and flow cytometric analyses demonstrated that hMSCs express FPR at the protein level. Actin polymerization after formylated peptide stimulation is one of the basic cellular responses indicative of the FPR function. We incubated hMSCs with 100nM of N-formyl methionyl leucyl phenylalanine (fMLP). Staining with phalloidin-conjugated fluorescein and flow cytometric analyses showed significant F-actin polymerization in hMSCs. Also, hMSCs were attracted toward fMLP gradients in Boyden chambers and binding of flourescein-labeled formyl-Nle-Leu-Phe-Nle-Tyr-Lys displayed the existence and functionality of the receptor in hMSCs. Thus, we conclude that hMSCs express functional formyl peptide receptors on their surface membrane and are capable of chemotaxis toward their ligand. The expression of the N-formyl peptide receptor on hMSCs, cells not normally associated with immune responses, could serve as a mechanism of directing stem cells to the sites of inflammation. Therefore, endogenous expression or over-expression of the receptor within hMSCs could be manipulated in a manner to target these cells to specific inflammatory sites, such as the lungs in cystic fibrosis.Funded in part by CFF and NIH. We thank Drs. Darwin J. Prockop and Bruce Bunnell at Tulane University for providing human hMSCs, technical support and advice. Human mesenchymal stem cells (hMSCs) from adult bone marrow can differentiate into a variety of different cell types including airway epithelial cells, suggesting the theraputic potential of stem cells in tissue injury repair. However, approaches for the efficient recruitmentof hMSCs to specific sites have not been established. To this end, we have investigated whether hMSCs express the N-formyl peptide receptor (FPR), which is typically expressed in polymorphonuclear leukocytes and is responsible for the neutrophil chemotactic response. Reverse transcription PCR using FPR specific primers resulted in a positive amplification of this mRNA. Sequencing of the amplicon proved 100% homology to the published FPR sequence. Immunofluorescence staining using a monoclonal antibody against human FPR and flow cytometric analyses demonstrated that hMSCs express FPR at the protein level. Actin polymerization after formylated peptide stimulation is one of the basic cellular responses indicative of the FPR function. We incubated hMSCs with 100nM of N-formyl methionyl leucyl phenylalanine (fMLP). Staining with phalloidin-conjugated fluorescein and flow cytometric analyses showed significant F-actin polymerization in hMSCs. Also, hMSCs were attracted toward fMLP gradients in Boyden chambers and binding of flourescein-labeled formyl-Nle-Leu-Phe-Nle-Tyr-Lys displayed the existence and functionality of the receptor in hMSCs. Thus, we conclude that hMSCs express functional formyl peptide receptors on their surface membrane and are capable of chemotaxis toward their ligand. The expression of the N-formyl peptide receptor on hMSCs, cells not normally associated with immune responses, could serve as a mechanism of directing stem cells to the sites of inflammation. Therefore, endogenous expression or over-expression of the receptor within hMSCs could be manipulated in a manner to target these cells to specific inflammatory sites, such as the lungs in cystic fibrosis. Funded in part by CFF and NIH. We thank Drs. Darwin J. Prockop and Bruce Bunnell at Tulane University for providing human hMSCs, technical support and advice.

Neutrophil differentiated HL‐60 cells model Mac‐1 (CD11b/CD18)‐independent neutrophil transepithelial migration

During active intestinal inflammation granulocytes accumulate in the lumen of the gut where they damage the epithelium through the release of various products such as reactive oxygen species and proteolytic enzymes. Previously, using function blocking monoclonal antibodies, we showed that neutrophil migration across intestinal epithelial monolayers in response to various chemoattractants was partially beta(2) integrin Mac-1 (CD11b/CD18)-independent. Here, we show that treating neutrophils with intact monoclonal antibody (mAb) to CD18 activates the cells to express more CD11b. Thus our goal now was to determine whether neutrophil Mac-1-independent transepithelial migration proceeds independently of prior cell activation through Mac-1. We took two approaches, one using blocking Fab' fragments of mAb to CD18 and the second was to develop a neutrophil differentiated HL-60 cell line which is Mac-1 deficient to further study neutrophil/epithelial cell interaction. Anti-CD18 Fab' minimally activated neutrophils but inhibited approximately 75% of transepithelial migration to fMLP while having a minimal effect (</=25% inhibition) on the migration to C5a. Upon incubation with dimethylsulphoxide, HL-60 cells differentiated and up-regulated CD11b expression and migrated to C5a and n-formyl methionyl leucyl phenylalanine in a similar manner to peripheral blood neutrophils. In contrast, CD11b expression was minimal on HL-60 cells differentiated with dibutytyl cAMP to a neutrophil-like phenotype. These cells, however, readily migrated across both intestinal and lung epithelial monolayers in response to C5a. We conclude that Mac-1-independent transepithelial migration does not require prior activation of cells via Mac-1 ligation because HL-60 cells lacking Mac-1 (CD11b/CD18) expression migrate effectively. HL-60 cells differentiated with dbcAMP should greatly assist in the search for the Mac-1-independent ligands for neutrophil migration across epithelium.

A new model for studying eosinophil migration across cultured intestinal epithelial monolayers.

Eosinophils play an important role in some gastrointestinal inflammatory conditions. Stimulated eosinophils migrate across the vascular endothelial wall and into the intestinal epithelium where by-products such as proteases may contribute to intestinal epithelial damage. Little is known about the epithelial migration of the eosinophils in the gut. The lack of data is attributable in part to the scarcity of human eosinophils for studies. HL-60-differentiated eosinophils present a means to perform studies on eosinophil function and chemotaxis. HL-60 clone 15 can be induced to differentiate into cells closely resembling human eosinophils. The authors describe a novel model for studying eosinophil migration across the intestinal epithelium. Fluorescent-labeled HL-60 eosinophils were incubated for 150 minutes on the basolateral surface of confluent and inverted T-84 monolayers separated by fluoroblock insert membranes. Chemotactic gradients of n-formyl methionyl leucyl phenylalanine (fMLP), eotaxin, and platelet aggregating factor (PAF) were used in variable concentrations. Changes in transepithelial electrical resistance (TEER) were compared with baseline values. Differentiated HL-60 eosinophils undergo migration in response to fMLP, PAF, and eotaxin. Migration is associated with a drop in TEER. In this model, HL-60-differentiated eosinophils migrate in response to stimulants chemotactic for human eosinophils. The transepithelial migration of eosinophils is associated with epithelial barrier dysfunction, which may contribute to the development of disease.

Neutrophil migration in inflammation: nitric oxide inhibits rolling, adhesion and induces apoptosis

There is controversy in the literature over whether nitric oxide (NO) released during the inflammatory process has a pro- or inhibitory effect on neutrophil migration. The aim of the present investigation was to clarify this situation. Treatment of rats with non-selective, N G-nitro- l-arginine (nitro), or selective inducible NO synthase (iNOS), aminoguanidine (amino) inhibitors enhanced neutrophil migration 6 h after the administration of low, but not high, doses of carrageenan (Cg) or Escherichia coli endotoxin (LPS). The neutrophil migration induced by N-formyl–methionyl–leucyl–phenylalanine (fMLP) was also enhanced by nitro or amino treatments. The enhancement of Cg-induced neutrophil migration by NOS inhibitor treatments was reversed by co-treatment with l-arginine, suggesting an involvement of the l-arginine/NOS pathway in the process. The administration of Cg in iNOS deficient (iNOS −/−) mice also enhanced the neutrophil migration compared with wild type mice. This enhancement was markedly potentiated by treatment of iNOS −/− mice with nitro. Investigating the mechanisms by which NOS inhibitors enhanced the neutrophil migration, it was observed that they promoted an increase in Cg-induced rolling and adhesion of leukocytes to endothelium and blocked the apoptosis of emigrated neutrophils. Similar results were observed in iNOS −/− mice, in which these mechanisms were potentiated and reverted by nitro and l-arginine treatments, respectively. In conclusion, these results suggest that during inflammation, NO released by either constitutive NOS (cNOS) or iNOS down-modulates the neutrophil migration. This NO effect seems to be a consequence of decreased rolling and adhesion of the neutrophils on endothelium and also the induction of apoptosis in migrated neutrophils.

Sputum chemotactic activity in chronic obstructive pulmonary disease: effect of α1–antitrypsin deficiency and the role of leukotriene B4 and interleukin 8

Background: Neutrophil recruitment to the airway is thought to be an important component of continuing inflammation and progression of chronic obstructive pulmonary disease (COPD), particularly in the presence of severe...

Differential Regulation of Neutrophil Phospholipase D Activity and Degranulation

One of the proposed functions of phosphatidic acid (PA) formation from phospholipase D (PLD) activation in neutrophils is to promote degranulation induced by receptor agonists. The present study shows that the time course and dose response of PA formation and degranulation induced by N-formyl–methionyl–leucyl-phenylalanine (fMLP) differed. PLD activation and degranulation also exhibited different dose response to genistein and epigallocatechin gallate (EGCG), inhibitors of protein tyrosine kinases. Genistein inhibited PLD activity with an IC50 value of 12.2 μM in fMLP- and 107 μM in phorbol myristate acetate (PMA)-stimulated cells. It required higher concentrations of genistein to inhibit degranulation than to inhibit PLD activity induced by fMLP. EGCG in the range of 40–400 μM had no effect on PLD activity but it inhibited the release of β-glucuronidase and elastase by fMLP-stimulated cells. These results demonstrate differential regulation of PLD activity and degranulation of primary granules by genistein and EGCG in fMLP-stimulated neutrophils.