PMN-like Cells Research Articles

Altered sialidase expression in human myeloid cells undergoing apoptosis and differentiation

To gain insight into sialic acid biology and sialidase/neuraminidase (NEU) expression in mature human neutrophil (PMN)s, we studied NEU activity and expression in PMNs and the HL60 promyelocytic leukemic cell line, and changes that might occur in PMNs undergoing apoptosis and HL60 cells during their differentiation into PMN-like cells. Mature human PMNs contained NEU activity and expressed NEU2, but not NEU1, the NEU1 chaperone, protective protein/cathepsin A(PPCA), NEU3, and NEU4 proteins. In proapoptotic PMNs, NEU2 protein expression increased > 30.0-fold. Granulocyte colony-stimulating factor protected against NEU2 protein upregulation, PMN surface desialylation and apoptosis. In response to 3 distinct differentiating agents, dimethylformamide, dimethylsulfoxide, and retinoic acid, total NEU activity in differentiated HL60 (dHL60) cells was dramatically reduced compared to that of nondifferentiated cells. With differentiation, NEU1 protein levels decreased > 85%, PPCA and NEU2 proteins increased > 12.0-fold, and 3.0-fold, respectively, NEU3 remained unchanged, and NEU4 increased 1.7-fold by day 3, and then returned to baseline. In dHL60 cells, lectin blotting revealed decreased α2,3-linked and increased α2,6-linked sialylation. dHL60 cells displayed increased adhesion to and migration across human bone marrow-derived endothelium and increased bacterial phagocytosis. Therefore, myeloid apoptosis and differentiation provoke changes in NEU catalytic activity and protein expression, surface sialylation, and functional responsiveness.

Intermediate Molecular Mass Hyaluronan and CD44 Receptor Interactions Enhance Neutrophil Phagocytosis and IL-8 Production via p38- and ERK1/2-MAPK Signalling Pathways.

CD44 is a common leukocyte adhesion molecule expressed on the surface of various cells. Hyaluronan (HA), the natural ligand of CD44, is a simple repeated disaccharide with variable molecular mass that is widely distributed on cell surfaces and the connective tissue matrix. The binding of small molecular mass HA (SMM-HA, MW<80kDa) to CD44 on immune-related cells elicits cell proliferation, differentiation, and cytokine production. However, the effects and molecular basis of intermediate molecular mass HA (IMM-HA, MW≈500kDa)-CD44 interactions on polymorphonuclear neutrophil (PMN) functions have not been elucidated. We hypothesised that IMM-HA would potentiate immune functions as well as SMM-HA. In the present study, we demonstrated IMM-HA and CD44 interactions enhanced normal PMN phagocytosis and IL-8 production compared to those with LPS or anti-CD45 treatment via F-actin cytoskeleton polymerization and subsequent ERK1/2- and p38-MAPK phosphorylation. Antibody-based inhibition of CD44 did not affect PMN function; however, F-actin aggregation was induced without MAPK phosphorylation. Enhanced PMN function via IMM-HA was determined to be CD44-dependent since this effect was abolished in DMSO-induced CD44(-) PMN-like cells obtained from HL-60 cells. In conclusion, we demonstrated that IMM-HA and CD44 interactions on PMNs potently elicit F-actin cytoskeleton polymerization and p38- and ERK1/2-MAPK phosphorylation to enhance PMN function.

Local application of adipose-derived mesenchymal stromal cells in experimental inflammatory OA results in interleukin-1β-mediated attraction of PMNs and reduced S100A8/A9 release

Purpose: Recent studies have demonstrated that mild synovitis in early phases of osteoarthritis (OA) is conducive to development of joint damage. OA synovitis is characterized by elevated levels of pro-inflammatory factors like S100A8, S100A9, interleukin-6 (IL-6), and interleukin-1 beta (IL-1β). S100A8/A9 was found to be crucial in mediating joint destruction in inflammatory experimental OA. Previously we found that adipose-derived mesenchymal stromal cells (ASCs) exhibit immunosuppressive characteristics and reduce joint pathology after local application into mouse knee joints with experimental inflammatory OA. This protective effect is only perceived after intra-articular injection in early but not late stage OA, suggesting that the effect may be mediated by an inflammatory environment. Our objective was to examine the working mechanism of ASCs after early injection in experimental OA. Methods: Experimental OA was induced by injection of collagenase into murine knee joints (CiOA). Total knee joints were stained with haematoxylin/eosin and the PMN-specific antibody NIMP-R14. ASCs were isolated from murine adipose tissue and stimulated for 24 hours with IL-1β or S100A8/A9. Gene expression in stimulated cells was analyzed using qPCR. Protein levels of chemokines and cytokines were measured in culture supernatant using Luminex. Migration of MACS isolated bone marrow (BM-) PMNs towards ASC-conditioned medium (CM) was examined using Transwell membranes. ASCs were co-cultured with BM-PMNs and analyzed using histology and Luminex. Results: Injection of ASCs into day 7 CiOA knee joints (when synovitis and IL-1β and S100A8/A9 levels are highest) caused a strong attraction of particularly PMN-like cells and their clustering around ASCs in the synovium shortly after injection (6 hours), which was confirmed by immunohistochemistry. IL-1β stimulation of ASCs in vitro strongly increased gene expression of PMN-attracting chemokines KC, CXCL5, and CXCL7 as well as protein levels of KC, whereas S100A8/A9 did not. The migration of BM-PMNs through Transwell membranes towards CM of IL-1β-stimulated ASCs was significantly increased (from 5% to 10%) when compared to CM of non-stimulated ASCs. Next, ASCs were co-cultured with BM-PMNs in the presence or absence of IL-1β. After 6 hours, a clear clustering of neutrophils around ASCs was observed, with a significant increase in the number of ASCs clustering with PMNs, as well as a significantly elevated number of clustering PMNs per ASC after IL-1β stimulation. Interestingly, association of PMNs with ASCs lead to a significantly diminished release of KC protein by ASCs (69% and 76% lower after 24 h and 48 h respectively), as well as a significantly reduced release of S100A8/A9 protein by the PMNs. This coincided with lowering of S100A8/A9 levels in washouts of inflamed synovium 6 h and 48 h after injection of ASCs in day 7 CiOA knee joints. Conclusions: Local application of ASCs in inflamed CiOA knee joints results in attraction and clustering of PMNs with ASCs in the synovium. This presumably runs via IL-1β-mediated up-regulation of chemokine release by ASCs, and ultimately results in significantly lowered levels of S100A8/A9. This research was supported by the Dutch Arthritis Association.

Salmonella enterica Serovar Typhimurium NiFe Uptake-Type Hydrogenases Are Differentially Expressed In Vivo

Salmonella enterica serovar Typhimurium, a common enteric pathogen, possesses three NiFe uptake-type hydrogenases. The results from mouse infection studies suggest that the H(2) oxidation capacity provided by these hydrogenases is important for virulence. Since the three enzymes are similar in structure and function, it may be expected that they are utilized under different locations and times during an infection. A recombination-based method to examine promoter activity in vivo (RIVET) was used to determine hydrogenase gene expression in macrophages, polymorphonuclear leukocyte (PMN)-like cells, and a mouse model of salmonellosis. The hyd and hya promoters showed increased expression in both murine macrophages and human PMN-like cells compared to that in the medium-only controls. Quantitative reverse transcription-PCR results suggested that hyb is also expressed in phagocytes. A nonpolar hya mutant was compromised for survival in macrophages compared to the wild type. This may be due to lower tolerance to acid stress, since the hya mutant was much more acid sensitive than the wild type. In addition, hya mutant cells were internalized by macrophages the same as wild-type cells. Mouse studies (RIVET) indicate that hyd is highly expressed in the liver and spleen early during infection but is expressed poorly in the ileum in infected animals. Late in the infection, the hyd genes were expressed at high levels in the ileum as well as in the liver and spleen. The hya genes were expressed at low levels in all locations tested. These results suggest that the hydrogenases are used to oxidize hydrogen in different stages of an infection.

On the influence of neutrophils in corneas with necrotizing HSV-1 keratitis following amniotic membrane transplantation

Necrotizing herpetic stromal keratitis (HSK) in mice rapidly improved after amniotic membrane transplantation (AMT). In this study we determined the fate of polymorphonuclear neutrophils (PMN) after AMT. AMT or tarsorrhaphy (T) was performed in BALB/c mice with ulcerative HSK. After 2 days, corneas were studied histologically and by transmission electron microscopy (TEM). CD11b, Gr-1, and TUNEL-positive cells were identified. Macrophages were depleted by subconjunctival injection of dichloromethylene-diphosphonate-liposomes (Cl 2MDP-LIP) before AMT. Corneas were studied for interleukin (IL)-1α, IL-2, interferon (IFN)-γ, CXCL1, CXCL2, and tumor necrosis factor (TNF)-α production by ELISA. PMN-enriched cell preparations co-cultured with amniotic membrane (AM) or with AM and such recombinant ( r) cytokines as rIL-1α, rIL-2, and rTNF-α or supernatants from activated lymphocytes were investigated by flow cytometry (Annexin-V/7-AAD and TUNEL), and a dimethylthiazolyl-diphenyltetrazolium-bromide (MTT)-viability assay. Corneas in the AMT mice had less inflammation, fewer PMN-like cells and fewer CD11b+, and Gr-1+ cells ( P < 0.01), but a higher ratio of apoptotic to viable PMN-resembling cells ( P < 0.01) than the T mice. Phagocytic removal of apoptotic PMN-like cells by macrophages was evident in the AMT group. After Cl 2MDP-LIP treatment, the corneas had more cell debris and apoptotic cells with PMN-like morphology. The concentrations of IL-1α, IL-2, CXCL1, and TNF-α were reduced in corneas of the AMT group as compared to that of the T group, while the concentration of CXCL2 was increased. Apoptosis of PMN-resembling cells was detected following cocultivation with AM, even when proinflammatory cytokines were present. Resolution of corneal inflammation in mice with necrotizing HSK after AMT is associated with increased apoptosis of PMN-like cells, reduction of pro-inflammatory cytokines, an increase of CXCL2, and increased removal of apoptotic PMN-like cells by macrophages.

Treatment of mice with the neutrophil-depleting antibody RB6-8C5 results in early development of experimental lyme arthritis via the recruitment of Gr-1- polymorphonuclear leukocyte-like cells.

Recently, we demonstrated that blocking the entry of neutrophils into Borrelia burgdorferi-infected joints in mice deficient in the chemokine receptor CXCR2 prevented the development of experimental Lyme arthritis. Neutrophils were marginalized in blood vessels at the site of infection but could not enter the joint tissue. In the present study, we treated both genetically arthritis-resistant DBA/2J (DBA) and arthritis-susceptible C3H/HeJ (C3H) mice with the neutrophil-depleting monoclonal antibody RB6-8C5 (RB6) to determine the effect on arthritis development. Surprisingly, both DBA and C3H mice treated with RB6 developed arthritis at 1 week postinfection, approximately 1 week earlier than the control-treated C3H mice. The early development of arthritis in the RB6-treated mice was accompanied by an influx into the joints of cells with ring-shaped polymorphonuclear leukocyte (PMN) cell morphology that were negative for the Gr-1 neutrophil maturation marker. RB6 treatment of mice also resulted in increased numbers of B. burgdorferi cells in the joints at 7 days postinfection and earlier expression of the chemokines KC and monocyte chemoattractant protein 1 in the joints compared to control-treated animals. Together, these results suggest that recruitment of neutrophils or PMN-like cells into an infected joint is a key requirement for Lyme arthritis development and that altered recruitment of these cells into the joints of arthritis-resistant mice can exacerbate the development of pathology.

Key role of proline-rich tyrosine kinase 2 in interleukin-8 (CXCL8/IL-8)-mediated human neutrophil chemotaxis.

The signalling pathways leading to CXCL8/IL-8-induced human neutrophil migration have not been fully characterized. The present study demonstrates that CXCL8 induces tyrosine phosphorylation as well as enzymatic activity of proline-rich tyrosine kinase 2 (Pyk2), a non-receptor protein tyrosine kinase (PTK), in human neutrophils. Induction of Pyk2 tyrosine phosphorylation by CXCL8 is regulated by Src PTK activation, whereas it is unaffected by phosphatidylinositol 3-kinase activation. Inhibition of Pyk2 activation by PP1, a Src PTK inhibitor, is paralleled by the inhibition of CXCL8-mediated neutrophil chemotaxis. Among CXCL8 receptors, Src protein tyrosine kinase activation selectively regulates CXCR1-mediated polymorphonuclear neutrophil (PMN) chemotaxis. Overexpression of PykM, the kinase-dead mutant of Pyk2, blocks CXCL8-induced chemotaxis of HL-60-derived PMN-like cells, thus pinpointing the key role of Pyk2 in CXCL8-induced chemotaxis.

The new ketolide HMR3647 accumulates in the azurophil granules of human polymorphonuclear cells.

HMR3647 is a semisynthetic representative of a new group of drugs, the ketolides, derived from erythromycin A. Since macrolides have been shown to accumulate in human polymorphonuclear cells (PMNs), we have investigated the ability of the molecule HMR3647 to enter human PMNs as well as other cell types, such as peripheral blood mononuclear cells and cell lines of hematopoietic and nonhematopoietic origin. In these experiments, HMR3647 was compared to erythromycin A, azithromycin, clarithromycin, and roxithromycin. Our results show that HMR3647 is specifically trapped in PMNs, where it is concentrated up to 300 times. In addition, it is poorly released by these cells, 80% of the compound remaining cell associated after 2 h in fresh medium. By contrast, it is poorly internalized and quickly released by the other cell types studied. This differs from the results obtained with the macrolide molecules, which behaved similarly in the different cells studied. In addition, subcellular fractionation of PMNs allowed us to identify the intracellular compartment where HMR3647 was trapped. In PMNs, more than 75% of the molecule was recovered in the azurophil granule fraction. Similarly, in NB4 cells differentiated into PMN-like cells, almost 60% of the molecules accumulated in the azurophil granule fraction. In addition, when HMR3647 was added to disrupted PMNs, 63% accumulated in the azurophil granules. Therefore, this study shows that the ketolide HMR3647 specifically accumulates in PMN azurophil granules, thus favoring its delivery to bacteria phagocytosed in these cells.

Regulation of ciprofloxacin uptake in human promyelocytic leukemia cells and polymorphonuclear leukocytes.

Polymorphonuclear leukocytes (PMNs) actively internalize ciprofloxacin, a capability that can enhance killing of intracellular bacteria and facilitate delivery of the antimicrobial agent to infection sites by migrating PMNs. In this study we investigated mechanisms for up-regulation of this process. Activation with N-formyl-methionyl-leucyl-phenylalanine (fMLP; 100 nM) enhanced PMN ciprofloxacin uptake by 50% (P < 0.05). Phorbol myristate acetate (PMA; > or = 10 nM) enhanced uptake by at least 36-fold, mainly by stimulating an increase in the Vmax of the ciprofloxacin transporter. This effect of PMA was inhibited by antagonists of protein kinase C (H7 and chelerythrine) and the mitogen-activated protein kinase cascade downstream (PD 098059). Under resting and PMA-activated conditions, ciprofloxacin uptake by immature human promyelocytic leukemia (HL-60) cells was much lower than in PMNs. However, when HL-60 cells were induced to mature into PMN-like cells, their ciprofloxacin uptake activity increased markedly. These findings implicate a role for protein kinase C in up-regulation of the ciprofloxacin transporter and suggest that myeloid cells acquire an enhanced ability to take up ciprofloxacin as they mature to end-stage PMNs.

Thrombospondin promotes both chemotaxis and haptotaxis in neutrophil-like HL-60 cells.

Differentiation of the human promyelocytic leukemia cell line HL-60 to polymorphonuclear leukocyte (PMN)-like cells with DMSO provides an important model for studying the acquisition of PMN functional responses that accompany differentiation. We showed previously that the extracellular matrix (ECM) protein thrombospondin (TSP) binds to PMN surface receptors and promotes adhesion and motility. Undifferentiated HL-60 cells did not adhere and were not motile in response to TSP, whereas cells differentiated toward PMN-like cells demonstrated both TSP-mediated adhesion and chemotaxis, with chemotaxis evident by day 2 of induction. With differentiation, a maximal response was obtained with 100 to 300 nM TSP, 10-fold lower than required for maximal PMN chemotaxis. Checkerboard analysis confirmed the directional nature of motility. mAb recognizing different domains of TSP inhibited chemotaxis, suggesting the involvement of multiple sites on TSP. Although both the NH2-terminal heparin-binding domain (HBD) and 140 kDa COOH-terminal fragment supported chemotaxis in PMN-like cells, neither fragment was as potent as intact TSP. Both pertussis and cholera toxin inhibited TSP-mediated chemotaxis, suggesting the involvement of GTP-binding proteins. The toxin effects did not indirectly result from elevated cAMP levels because high concentrations of either 8-bromo-cAMP or dibutyryl cAMP did not inhibit chemotaxis. TSP bound to nitrocellulose filters induced the directed migration (haptotaxis) of PMN-like cells rather than the random motility observed with PMN. Haptotaxis was stimulated by either the HBD or 140-kDa fragment and was inhibited by mAb against these two domains. Haptotaxis rather than random migration was confirmed by checkerboard analysis. Our results demonstrate that PMN-like HL-60 cells respond differently to TSP than human peripheral blood PMN. These differences may reflect 1) an aberration in HL-60 differentiation reflecting their leukemic phenotype 2) differentiation of HL-60 cells to a cell type characteristic of "activated" PMN.