Primary Leukocyte Cultures Research Articles

Ubiquitin receptor binding and signaling in primary human leukocytes

Utilizing the human monocyte/macrophage cell line THP1, we recently identified extracellular ubiquitin as an endogenous agonist of the G protein-coupled receptor CXC chemokine receptor (CXCR) 4. Because receptor binding and signaling properties of extracellular ubiquitin have not been evaluated in primary human leukocytes, we analyzed its binding characteristics and subsequent Ca2+ signaling in freshly isolated human B-cells, T-cells and monocytes. Ubiquitin binding shows typical receptor binding characteristics and promotes intracellular Ca2+ flux within seconds in all three cell populations. The Kd for the ubiquitin receptor interaction in freshly isolated human monocytes is consistent with the affinity of the ubiquitin CXCR4 interaction that we reported for THP1 cells. As detected in THP1 cells previously, the ubiquitin induced Ca2+ flux can be attenuated with a phospholipase C inhibitor in all primary leukocyte cultures. Our observations further support the finding that ubiquitin is a CXCR4 agonist and demonstrate that extracellular ubiquitin induces physiological relevant signaling events in primary human leukocytes. Although the exact mechanism of the ubiquitin CXCR4 interaction, its receptor selectivity and subsequent signaling events remain to be determined, our findings identify a novel and unexpected biological role of extracellular ubiquitin as an endogenous immune modulator.

Cooperative and Synergistic Activities of the Epithelial Cell Derived Cytokines TSLP, IL-25, and IL-33 on Primary Leukocyte Cultures

RATIONALE: The cytokines thymic stromal lymphopoietin (TSLP), IL-25, and IL-33 individually have been shown to have a profound influence on the development of allergic inflammatory responses. The common epithelial cell origin of these cytokines and overlapping target cell populations suggest that in an appropriate inflammatory state the opportunity for cooperative activities between TSLP, IL-25, and IL-33 may exist. Utilizing a variety of primary human and mouse leukocyte cultures and readouts we examined the individual and combined effects of TSLP, IL-25, and IL-33.

Antioxidant/prooxidant activity of a polyphenolic grape seed extract

Abstract The oxidative potential of a polyphenolic grape seed extract, with the idea of using this extract as a nutritive supplement, was evaluated. Data presented in this work provide in vitro (primary leukocyte culture) UV–Vis spectral evidence, indicating that quinones, as oxidation products, are involved in the modulation of the antioxidant/prooxidant balance at cellular level in the case of catechin-type compounds (pure catechin (CS) and polyphenolic extract (PE)), in the absence or presence of lipoxygenase (pure (LS) or in raw soybean extract (LE)) as oxidative stress inducers. The study shows, to some extent, the grape seed extract tested, considered as an antioxidant nutritive supplement, may have prooxidant activity as well, depending on the dose, duration of administration, and other dietary components. The UV–Vis analysis confirms that the antioxidant activity of this extract might be mediated by prooxidant quinones and oxidation products of the polyphenols from grape seeds.

Interferon type I and type II responses in an Atlantic salmon (Salmo salar) SHK-1 cell line by the salmon TRAITS/SGP microarray

Interferons (IFNs) are cytokines that have proinflammatory, antiviral, and immunomodulatory effects and play a central role during a host response to pathogens. The IFN family contains both type I and type II molecules. While there are a number of type I IFNs, there is only one type II IFN. Recently both type I and type II IFN genes have been cloned in salmonid fish and recombinant proteins produced showing IFN activity. We have stimulated an Atlantic salmon cell line (SHK-1) with both type I and type II recombinant salmonid IFNs and analyzed the transcriptional response by microarray analysis. Cells were exposed to recombinant IFNs for 6 or 24 h or left unexposed as controls. RNA was hybridized to an Atlantic salmon cDNA microarray (salmon 17K feature TRAITS/SGP array) in order to assess differential gene expression in response to IFN exposure. For IFN I and II, 47 and 72 genes were stimulated, respectively; most genes were stimulated by a single IFN type, but some were affected by both IFNs, indicating coregulation of the IFN response in fish. Real-time PCR analysis was employed to confirm the microarray results for selected differentially expressed genes in both a cell line and primary leukocyte cultures.

Induction of inflammatory cytokines by cartilage extracts

Shark cartilage extracts were examined for induction of cytokines and chemokines in human peripheral blood leukocytes. Primary leukocyte cultures were exposed to a variety of aqueous and organic extracts prepared from several commercial brands of shark cartilage. From all commercial sources of shark cartilage tested the acid extracts induced higher levels of TNFα than other extracts. Different commercial brands of shark cartilage varied significantly in cytokine-inducing activity. TNFα induction was seen as early as 4 h and IFNγ at detectable levels for up to four days. Shark cartilage extracts did not induce physiologically significant levels of IL-4. Results suggest that shark cartilage, preferentially, induces Th1 type inflammatory cytokines. When compared to bovine cartilage extract, collagen, and chondroitin sulfate, shark cartilage induced significantly higher levels of TNFα. Treatment with digestive proteases (trypsin and chymotrypsin) reduced the cytokine induction response by 80%, suggesting that the active component(s) in cartilage extracts is proteinaceous. The induction of Th1 type cytokine response in leukocytes is a significant finding since shark cartilage, taken as a dietary supplement for a variety of chronic degenerative diseases, would be contraindicated in cases where the underlying pathology of the chronic condition is caused by inflammation.

Exploring the pharmacology of the leukotriene B 4 receptor BLT 1, without the confounding effects of BLT 2

Most previous studies of leukotriene B 4 (LTB 4) pharmacology using primary leukocyte cultures and myeloid cell lines do not differentiate between leukotriene BLT 1 and BLT 2 receptor activation because both receptors are often expressed by these cells. Here we show that in HeLa cells expressing BLT 1 but not BLT 2 receptors, BLT 1 receptor activation resulted in IP 3 mediated calcium release from intracellular stores initially, followed by calcium influx through cell membrane channels. BLT 1 calcium signalling was sensitive to the activity of protein kinase C (PKC), protein kinase A (PKA) and protein-tyrosine kinases (PTKs), as well as changes in membrane cholesterol levels and treatments that are known to disrupt normal membrane physiology and/or lipid rafts. Inhibition of MAP kinases, Rho-associated kinases, or phosphoinositol-3-kinases (PI 3K) had no effect on BLT 1 receptor induced calcium signalling, and the receptor was insensitive to the redox state of the extracellular compartment.

6] Induction and production of human interferon with human leukemic cells

Publisher Summary Patients with chronic myelogenous leukemia (CML) and elevated white cell counts afford a large number of leukocytes for production of interferon. Primary leukocyte cultures are established in a synthetic medium with casein instead of the serum requirement and are induced with Newcastle disease virus to produce interferon. Procedures involved in the establishment of the primary culture are performed with aseptic techniques. Equipment is steam-sterilized, heat treated at 230 ° or rinsed with denatured ethyl alcohol (3A) depending on the applicability. Whenever possible, procedures are performed in a laminar-flow hood. Cells are obtained in blood collection bags after leukapheresis of chronic myelogenous leukemia patients. Titers of interferon from leukemic leukocytes can be as high as 200,000 units/ml. A quantitative recovery of interferon activity is obtained for the procedures outlined.

Studies on the Contact Sensitization of Man with Simple Chemicals II. Lymphokine Production in Allergic Contact Dermatitis to Dinitrochlorobenzene

A cell-free soluble factor(s) was specifically generated in primary leukocyte cultures during interaction of lymphocytes from a dinitrochlorobenzene (DNCB)-sensitized subject with DNCB-erythrocyte complexes (DNCB-antigen). The factor(s) was capable of inducing blastogenesis and DNA synthesis in secondary autologous DNCB-sensitive leukocyte cultures and in leukocyte cultures of allogeneic subjects who had not been sensitized to DNCB. Under the culture conditions employed to generate the factor(s), maximal DNA synthesis was induced in secondary insensitive cultures by supernates collected after 1–3 days of primary culture. Higher concentrations of primary stimulated culture supernates induced greater degrees of DNA synthesis in secondary insensitive leukocyte cultures with instances of greater than 100-fold stimulation. The discovery of lymphokine activity in experimentally induced allergic contact dermatitis offers a new and advantageous approach for investigating the biologic significance of these substances and may assist in better understanding cell-mediated immunity and the relationship of allergic contact dermatitis to other forms of delayed type hypersensitivity.