Leslie C. McKinney

Patch-Clamp Studies in Human Macrophages: Single-Channel and Whole-Cell Characterization of Two K § Conductances

SummaryHuman peripheral blood monocytes cultured for varying periods of time were studied using whole-cell and single-channel patch-clamp recording techniques. Whole-cell recordings revealed both an outward K current activating at potentials >20 mV and an inwardly rectifying K current present at potentials negative to −60 mV. Tail currents elicited by voltage steps that activated outward current reversed nearEK, indicating that the outward current was due to a K conductance. TheI–V curve for the macroscopic outward current was similar to the mean single-channelI–V curve for the large conductance (240 pS in symmetrical K) calcium-activated K channel present in these cells. TEA and charybdotoxin blocked the whole-cell outward current and the single-channel current. Excised and cell-attached single-channel data showed that calcium-activated K channels were absent in freshly isolated monocytes but were present in >85% of patches from macrophages cultured for >7 days. Only 35% of the human macrophages cultured for >7 days exhibited whole-cell inward currents. The inward current was blocked by external barium and increased when [K]o increased. Inward-rectifying single-channel currents with a conductance of 28 pS were present in cells exhibiting inward whole-cell currents. These single-channel currents are similar to those described in detail in J774.1 cells (L.C. McKinney & E.K. Gallin,J. Membrane Biol.103:41–53, 1988).

Effect of Adherence, Cell Morphology, and Lipopolysaccharide on Potassium Conductance and Passive Membrane Properties of Murine Macrophage J774. 1 Cells

SummaryThe effects of adherence, cell morphology, and lipopolysaccharide on electrical membrane properties and on the expression of the inwardly rectifying K conductance in J774.1 cells were investigated. Whole-cell inwardly rectifying K currents (Ki), membrane capacitance (Cm), and membrane potential (Vm) were measured using the patch-clamp technique. SpecificKi conductance (GKi, whole-cell Ki conductance corrected for leak and normalized to membrane capacitance) was measured as a function of time after adherence, and was found to increase almost twofold one day after plating. Membrane potential (Vm) also increased from −42±4 mV (n=32) to −58±2 mV (n=47) over the same time period.GKi andVm were correlated with each other;GL (leak conductance normalized to membrane capacitance) andVm were not. The magnitudes ofGKi andVm 15 min to 2 hr after adherence were unaffected by the presence of 100 μm cycloheximide, but the increase inGKiandVm that normally occurred between 2 and 8 hr after adherence was abolished by cycloheximide treatment. Membrane properties were analyzed as a function of cell morphology, by dividing cells into three categories ranging from small round cells to large, extremely spread cells. The capacitance of spread cells increased more than twofold within one day after adherence, which indicates that spread cells inserted new membrane. Spread cells had more negative resting membrane potentials than round cells, butGKi andGL were not significantly different. Lipopolysaccharide-(LPS; 1 or 10 μg/ml) treated cells showed increasedCm compared to control cells plated for comparable times. In contrast to the effect of adherence, LPS-treated cells exhibited a significantly lowerGKi than control cells, indicating that the additional membrane did not have as high a density of functionalGKi channels. We conclude that both adherence and LPS treatment increase the total surface membrane area of J774 cells and change the density of Ki channels. In addition, this study demonstrates that membrane area and density of Ki channels can vary independently of one another.

Astrocytes as targets for Venezuelan equine encephalitis virus infection

Venezuelan equine encephalitis virus (VEE) produces an acute infection in humans and induces a well-characterized cytopathic effect in neurons of the central nervous system (CNS). However, little is known about the role of glial cells in response to VEE infection of the CNS. Our results demonstrate that VEE is capable of a productive infection in primary astrocyte cultures and that this infection is cytotoxic. Further, there were significant differences in the growth kinetics comparing virulent and attenuated strains of VEE. Additionally, VEE infection of astrocyte cultures induced gene expression of two neuro-immune modulators, tumor necrosis factor-alpha (TNF-alpha) and inducible nitric oxide synthase (iNOS). Assays for TNF-alpha protein and nitric oxide (NO) demonstrated high levels of TNF-alpha protein and low levels of NO in response to VEE infection of astrocytes. These observations suggest an important role of astrocytes in this virus-induced encephalitis, and that interactions between astrocytes, other glial cells, and neurons may be important in VEE pathogenesis. Such interactions, which could impact neuronal survival, may include loss of functional changes in astrocytes or, alternatively, their production of neurotoxic molecules.

Ionizing radiation potentiates the induction of nitric oxide synthase by IFN-gamma and/or LPS in murine macrophage cell lines: role of TNF-alpha.

Macrophages are activated to become cytotoxic by a highly coordinated set of cytokine signals. Ionizing radiation can mimic cytokine signals and lead to enhanced states of activation. We tested the ability of γ‐radiation, alone and with interferon‐γ (IFN‐γ) and/or lipopolysaccharide (LPS), to induce nitric oxide (NO) production in J774.1 and RAW264.7 murine macrophages. NO was induced weakly, moderately, or strongly by IFN‐γ alone, LPS alone, or IFN‐γ + LPS, respectively. Radiation alone (0.5–50 Gy) did not induce NO, but enhanced NO production in a dose‐dependent manner (0.5–5 Gy) when cells were exposed to IFN‐γ or LPS 24 h post‐irradiation. Immunoblots showed parallel induction of nitric oxide synthase (NOS2). Application of antitumor necrosis factor α (TNF‐α) antibody before irradiation blocked induction of NO by IFN‐γ. We conclude (1) that irradiated cells produce more NO in response to either IFN‐γ or LPS and (2) that the increase is mediated by induction of TNF‐α. J. Leukoc. Biol. 64: 459–466; 1998.

Ionizing Radiation Potentiates the Induction of Nitric Oxide Synthase by Interferon‐γ and/or Lipopolysaccharide in Murine Macrophage Cell Lines: Role of Tumor Necrosis Factor‐α

Abstract: Macrophages respond to infection or injury by changing from a “resting” cellular phenotype to an “activated” state defined by the expression of various cytotoxic effector functions. Regulation of the transition from a resting to an activated state is effected by cytokine and/or pathogenic signals. Some signals do not directly induce activation, but instead “prime” the macrophage to respond more vigorously to a second signal. One example of this priming phenomenon involves induction of nitric oxide (NO) synthesis by the enzyme nitric oxide synthase (NOS2). Our experiments indicate that low doses (1–5 Gy) of ionizing radiation can enhance the induction of enzymatically active NOS2 by IFN‐γ or LPS in J774.1 and RAW264.7 murine macrophage cell lines. Radiation alone did not produce this induction, rather, it was effective as a priming signal; cells exposed to radiation produced more NO when a second signal, either IFN‐γ or LPS, was applied 24 h later.

Induction of nitric oxide production in infiltrating leukocytes following in vivo irradiation of tumor-bearing mice†

Nitric oxide (NO) has been implicated both in regression and progression of tumors due to its production by both tumor cells and infiltrating leukocytes. Ionizing radiation causes the regression of tumors, and can augment the production of NO by macrophages in vitro. We examined the cellular and systemic production of NO in mice in which radiation-resistant RIF-1 fibrosarcoma cells were implanted subcutaneously and were then either irradiated or sham-treated at the tumor site. Ten days following implantation of the tumors, CD45- tumor cells and CD45+ leukocytes were derived from resected tumors immediately after irradiation with 60 Gy, a dose previously reported to reduce tumor growth. Leukocytes from tumors of irradiated hosts produced spontaneously up to four-fold more NO than did either leukocytes from unirradiated mice or CD45- tumor cells from either unirradiated or irradiated mice. Between days 10-14 following tumor implantation, serum NO2-/NO3- increased in both irradiated and unirradiated mice to an equal extent, culminating in levels higher than those of non-tumor-bearing mice. Though NO production is elevated in macrophages treated with 1-10 Gy of radiation in vitro, higher doses may be required by tumor-infiltrating macrophages in vivo and thus may indicate that tumor-infiltrating macrophages are deactivated.

Modulation of protein expression and activity by radiation: Relevance to intracoronary radiation for the prevention of restenosis

Restenosis is a common complication of percutaneous transluminal coronary angioplasty. Recent studies have demonstrated a striking reduction in the neointimal hyperplasia characteristic of restenosis following intracoronary radiation (IR), but the mechanisms by which radiation reduces neointima formation following balloon overstretch injury are not elucidated fully. In addition to direct antimitotic effects mediated via oxygen free radicals, ionizing radiation can induce the expression of numerous genes and thereby mediate indirect effects. Additionally, IR prevents restenosis at the cost of decreased healing and increased thrombosis, and we suggest that these adverse reactions can be modulated by adjunct pharmacology or gene-based strategies. This review discusses several genes and proteins modulated by radiation in the context of arterial injury, and their possible therapeutic relevance.

Chapter 6 Monovalent Ion Transport and Membrane Potential Changes during Activation in Phagocytic Leukocytes

Publisher Summary This chapter discusses the ionic basis for the resting membrane potential in the neutrophil and the macrophage; properties of the different types of ionic channels, pumps, and carriers that are present in these cells; and the possible role of membrane potential and ionic conductances in phagocyte function. The resting membrane potentials of both neutrophils and macrophages are known, and a number of the ion channels and carriers in the phagocyte membrane have been characterized. In the macrophage, several K channels that are voltage or calcium sensitive have been described as well as a large conductance chloride channel and cation channel activated by ligand binding. Voltage-dependent calcium and sodium channels have not been found in either neutrophils or macrophages. It is possible that calcium channels similar to those described in human T lymphocytes also exist in phagocytes but that they have not been detected to date because of their very small single-channel conductance and activation by second messengers rather than by voltage.