Neutrophils: the forgotten cell in JIA disease pathogenesis

Juvenile idiopathic arthritis (JIA) has long been assumed to be an autoimmune disease, triggered by aberrant recognition of "self" antigens by T-cells. However, systems biology approaches to this family of diseases have suggested complex interactions between innate and adaptive immunity that underlie JIA. In particular, new data suggest an important role for neutrophils in JIA pathogenesis. In this short review, we will discuss the new data that support a role for neutrophils in JIA, discuss regulatory functions that link neutrophils to adaptive immune responses, and discuss future areas of investigation. Above all else, we invite the reader to re-consider the use of the term "autoimmunity" as applied to the family of illnesses we collectively call JIA

Reactive oxygen species in phagocytic leukocytes

Phagocytic leukocytes consume oxygen and generate reactive oxygen species in response to appropriate stimuli. The phagocyte NADPH oxidase, a multiprotein complex, existing in the dissociated state in resting cells becomes assembled into the functional oxidase complex upon stimulation and then generates superoxide anions. Biochemical aspects of the NADPH oxidase are briefly discussed in this review; however, the major focus relates to the contributions of various modes of microscopy to our understanding of the NADPH oxidase and the cell biology of phagocytic leukocytes

Sulfhydryl Modification Induces Calcium Entry through IP3-Sensitive Store-Operated Pathway in Activation-Dependent Human Neutrophils

As the first line of host defense, neutrophils are stimulated by pro-inflammatory cytokines from resting state, facilitating the execution of immunomodulatory functions in activation state. Sulfhydryl modification has a regulatory role in a wide variety of physiological functions through mediation of signaling transductions in various cell types. Recent research suggested that two kinds of sulfhydryl modification, S-nitrosylation by exogenous nitric oxide (NO) and alkylation by N-ethylmaleimide (NEM), could induce calcium entry through a non-store-operated pathway in resting rat neutrophils and DDT1MF-2 cells, while in active human neutrophils a different process has been observed by us. In the present work, data showed that NEM induced a sharp rising of cytosolic calcium concentration ([Ca2+]c) without external calcium, followed by a second [Ca2+]c increase with readdition of external calcium in phorbol 12-myristate 13-acetate (PMA)-activated human neutrophils. Meanwhile, addition of external calcium did not cause [Ca2+]c change of Ca2+-free PMA-activated neutrophils before application of NEM. These data indicated that NEM could induce believable store-operated calcium entry (SOCE) in PMA-activated neutrophils. Besides, we found that sodium nitroprusside (SNP), a donor of exogenous NO, resulted in believable SOCE in PMA-activated human neutrophils via S-nitrosylation modification. In contrast, NEM and SNP have no effect on [Ca2+]c of resting neutrophils which were performed in suspension. Furthermore, 2-Aminoethoxydiphenyl borate, a reliable blocker of SOCE and an inhibitor of inositol 1,4,5-trisphosphate (IP3) receptor, evidently abolished SNP and NEM-induced calcium entry at 75 µM, while preventing calcium release in a concentration-dependent manner. Considered together, these results demonstrated that NEM and SNP induced calcium entry through an IP3-sensitive store-operated pathway of human neutrophils via sulfhydryl modification in a PMA-induced activation-dependent manner

Ion channel clustering enhances weak electric field detection by neutrophils: apparent roles of SKF96365-sensitive cation channels and myeloperoxidase trafficking in cellular responses

We have tested Galvanovskis and Sandblom’s prediction that ion channel clustering enhances weak electric field detection by cells as well as how the elicited signals couple to metabolic alterations. Electric field application was timed to coincide with certain known intracellular chemical oscillators (phase-matched conditions). Polarized, but not spherical, neutrophils labeled with anti-K v 1.3, FL-DHP, and anti-TRP1, but not anti-T-type Ca 2+ channels, displayed clusters at the lamellipodium. Resonance energy transfer experiments showed that these channel pairs were in close proximity. Dose-field sensitivity studies of channel blockers suggested that K + and Ca 2+ channels participate in field detection, as judged by enhanced oscillatory NAD(P)H amplitudes. Further studies suggested that K + channel blockers act by reducing the neutrophil’s membrane potential. Mibefradil and SKF93635, which block T-type Ca 2+ channels and SOCs, respectively, affected field detection at appropriate doses. Microfluorometry and high-speed imaging of indo-1-labeled neutrophils was used to examine Ca 2+ signaling. Electric fields enhanced Ca 2+ spike amplitude and triggered formation of a second traveling Ca 2+ wave. Mibefradil blocked Ca 2+ spikes and waves. Although 10 μM SKF96365 mimicked mibefradil, 7 μM SKF96365 specifically inhibited electric field-induced Ca 2+ signals, suggesting that one SKF96365-senstive site is influenced by electric fields. Although cells remained morphologically polarized, ion channel clusters at the lamellipodium and electric field sensitivity were inhibited by methyl-β-cyclodextrin. As a result of phase-matched electric field application in the presence of ion channel clusters, myeloperoxidase (MPO) was found to traffic to the cell surface. As MPO participates in high amplitude metabolic oscillations, this suggests a link between the signaling apparatus and metabolic changes. Furthermore, electric field effects could be blocked by MPO inhibition or removal while certain electric field effects were mimicked by the addition of MPO to untreated cells. Therefore, channel clustering plays an important role in electric field detection and downstream responses of morphologically polarized neutrophils. In addition to providing new mechanistic insights concerning electric field interactions with cells, our work suggests novel methods to remotely manipulate physiological pathways.Peer Reviewedhttp://deepblue.lib.umich.edu/bitstream/2027.42/46726/1/249_2005_Article_1.pd

Computer Aided Identification of Small Molecules Disrupting uPAR/α5β1- Integrin Interaction: A New Paradigm for Metastasis Prevention

Disseminated dormant cancer cells can resume growth and eventually form overt metastases, but the underlying molecular mechanism responsible for this change remains obscure. We previously established that cell surface interaction between urokinase receptor (uPAR) and alpha5beta1-integrin initiates a sequel of events, involving MAPK-ERK activation that culminates in progressive cancer growth. We also identified the site on uPAR that binds alpha5beta1-integrin. Disruption of uPAR/integrin interaction blocks ERK activation and forces cancer cells into dormancy.Using a target structure guided computation docking we identified 68 compounds from a diversity library of 13,000 small molecules that were predicted to interact with a previously identified integrin-binding site on uPAR. Of these 68 chemical hits, ten inhibited ERK activation in a cellular assay and of those, 2 compounds, 2-(Pyridin-2-ylamino)-quinolin-8-ol and, 2,2'-(methylimino)di (8-quinolinol) inhibited ERK activation by disrupting the uPAR/integrins interaction. These two compounds, when applied in vivo, inhibited ERK activity and tumor growth and blocked metastases of a model head and neck carcinoma.We showed that interaction between two large proteins (uPAR and alpha5beta1-integrin) can be disrupted by a small molecule leading to profound downstream effects. Because this interaction occurs in cells with high uPAR expression, a property almost exclusive to cancer cells, we expect a new therapy based on these lead compounds to be cancer cell specific and minimally toxic. This treatment, rather than killing disseminated metastatic cells, should induce a protracted state of dormancy and prevent overt metastases

Scavenger receptors and β-glucan receptors participate in the recognition of yeasts by murine macrophages

Objectives: Numerous receptors have been implicated in recognition of pathogenic fungi by macrophages, including the $\beta$-glucan receptor dectin-1. The role of scavenger receptors (SRs) in anti-fungal immunity is not well characterized. Methods: We studied uptake of unopsonized Saccharomycetes cerevisiae (zymosan) and live Candida albicans yeasts as well as zymosan-stimulated $H_2O_2$ production in J774 macrophage-like cells and peritoneal exudate macrophages (PEMs). The role of different receptors was assessed with the use of competitive ligands, transfected cells and receptor-deficient macrophages. Results: The uptake of zymosan by untreated J774 cells was mediated approximately half by SRs and half by a $\beta$-glucan receptor which was distinct from dectin-1 and not linked to stimulation of $H_2O_2$ production. Ligands of $\beta$-glucan receptors and of SRs also inhibited uptake of C. albicans by macrophages (J774 cells and PEMs). In macrophages pretreated with a CpG motif-containing oligodeoxynucleotide (CpG-ODN) the relative contribution of SRs to yeast uptake increased and that of $\beta$-glucan receptors decreased. Whereas the class A SR MARCO participated in the uptake of both zymosan and C. albicans by CpG-ODN-pretreated, but not untreated macrophages, the related receptor SR-A/CD204 was involved in the uptake of zymosan, but not of C. albicans. The reduction of zymosan-stimulated $H_2O_2$ production observed in DS-pretreated J774 cells and in class A SRs-deficient PEMs suggest that class A SRs mediate part of this process. Conclusions: Our results revealed that SRs belong to a redundant system of receptors for yeasts. Binding of yeasts to different receptors in resting versus CpG-ODN-pre-exposed macrophages may differentially affect polarization of adaptive immune responses

Gemcitabine with a specific conformal 3D 5FU radiochemotherapy technique is safe and effective in the definitive management of locally advanced pancreatic cancer

The aim of this phase II study was to assess the feasibility and efficacy of a specific three-dimensional conformal radiotherapy technique with concurrent continuous infusion of 5-fluorouracil (CI 5FU) sandwiched between gemcitabine chemotherapy in patients with locally advanced pancreatic cancer. Patients with inoperable cancer in the pancreatic head or body without metastases were given gemcitabine at 1000 mg m−2 weekly for 3 weeks followed by a 1-week rest and a 6-week period of radiotherapy and concurrent CI 5FU (200 mg m−2 day−1). The defined target volume was treated to 54 Gy in 30 daily fractions of 1.8 Gy. After 4 weeks' rest, gemcitabine treatment was re-initiated for three cycles (days 1, 8, 15, q28). Forty-one patients were enrolled. At the end of radiotherapy, one patient (2.4%) had a complete response and four patients (9.6%) had a partial response; at the end of treatment, three patients (7.3%) had a complete response and two patients (4.9%) had a partial response. Median survival time was 11.7 months, median time to progression was 7.1 months, and median time to failure of local control was 11.9 months. The 1- and 2-year survival rates were 46.3 and 9.8%, respectively. Treatment-related grade 3 and 4 toxicities were reported by 16 (39.0%) and four (9.8%) patients, respectively. Sixteen out of 41 patients did not complete the planned treatment and nine due to disease progression. This approach to treatment of locally advanced pancreatic cancer is safe and promising, with good local control for a substantial proportion of patients, and merits testing in a randomised trial

A Third Measure-Metastable State in the Dynamics of Spontaneous Shape Change in Healthy Human's White Cells

Human polymorphonuclear leucocytes, PMN, are highly motile cells with average 12-15 µm diameters and prominent, loboid nuclei. They are produced in the bone marrow, are essential for host defense, and are the most populous of white blood cell types. PMN also participate in acute and chronic inflammatory processes, in the regulation of the immune response, in angiogenesis, and interact with tumors. To accommodate these varied functions, their behavior is adaptive, but still definable in terms of a set of behavioral states. PMN morphodynamics have generally involved a non-equilibrium stationary, spheroid Idling state that transitions to an activated, ellipsoid translocating state in response to chemical signals. These two behavioral shape-states, spheroid and ellipsoid, are generally recognized as making up the vocabulary of a healthy PMN. A third, “random” state has occasionally been reported as associated with disease states. I have observed this third, Treadmilling state, in PMN from healthy subjects, the cells demonstrating metastable dynamical behaviors known to anticipate phase transitions in mathematical, physical, and biological systems. For this study, human PMN were microscopically imaged and analyzed as single living cells. I used a microscope with a novel high aperture, cardioid annular condenser with better than 100 nanometer resolution of simultaneous, mixed dark field and intrinsic fluorescent images to record shape changes in 189 living PMNs. Relative radial roundness, R(t), served as a computable order parameter. Comparison of R(t) series of 10 cells in the Idling and 10 in the Treadmilling state reveals the robustness of the “random” appearing Treadmilling state, and the emergence of behaviors observed in the neighborhood of global state transitions, including increased correlation length and variance (divergence), sudden jumps, mixed phases, bimodality, power spectral scaling and temporal slowing. Wavelet transformation of an R(t) series of an Idling to Treadmilling state change, demonstrated behaviors concomitant with the observed transition