Daniel J. Loegering

Differential Requirement for Classic and Novel PKC Isoforms in Respiratory Burst and Phagocytosis in RAW 264.7 Cells

The binding of Ab (IgG)-opsonized particles by FcγRs on macrophages results in phagocytosis of the particles and generation of a respiratory burst. Both IgG-stimulated phagocytosis and respiratory burst involve activation of protein kinase C (PKC). However, the specific PKC isoforms required for these responses have yet to be identified. We have studied the involvement of PKC isoforms in IgG-mediated phagocytosis and respiratory burst in the mouse macrophage-like cell line, RAW 264.7. Like primary monocyte/macrophages, their IgG-mediated phagocytosis was calcium independent and diacylglycerol sensitive, consistent with novel PKC activation. Respiratory burst in these cells was Ca2+ dependent and inhibited by staurosporine and calphostin C as well as by the classic PKC-selective inhibitors Gö 6976 and CGP 41251, suggesting that classic PKC is required. In contrast, phagocytosis was blocked by general PKC inhibitors but not by the classic PKC-specific drugs. RAW 264.7 cells expressed PKCs α, βI, δ, ε, and ζ. Subcellular fractionation demonstrated that PKCs α, δ, and ε translocate to membranes during phagocytosis. In Ca2+-depleted cells, only novel PKCs δ and ε increased in membranes, and the time course of their translocation was consistent with phagosome formation. Confocal microscopy of cells transfected with green fluorescent protein-conjugated PKC α or ε confirmed that these isoforms translocated to the forming phagosome in Ca-replete cells, but only PKC ε colocalized with phagosomes in Ca2+-depleted cells. Taken together, these results suggest that the classic PKC α mediates IgG-stimulated respiratory burst in macrophages, whereas the novel PKCs δ and/or ε are necessary for phagocytosis.

Protein Kinase C and Toll-Like Receptor Signaling

Protein kinase C (PKC) is a family of kinases that are implicated in a plethora of diseases, including cancer and cardiovascular disease. PKC isoforms can have different, and sometimes opposing, effects in these disease states. Toll-like receptors (TLRs) are a family of pattern recognition receptors that bind pathogens and stimulate the secretion of cytokines. It has long been known that PKC inhibitors reduce LPS-stimulated cytokine secretion by macrophages, linking PKC activation to TLR signaling. Recent studies have shown that PKC-α, -δ, -ε, and -ζ are directly involved in multiple steps in TLR pathways. They associate with the TLR or proximal components of the receptor complex. These isoforms are also involved in the downstream activation of MAPK, RhoA, TAK1, and NF-κB. Thus, PKC activation is intimately involved in TLR signaling and the innate immune response.

A role for PKC-ε in FcγR-mediated phagocytosis by RAW 264.7 cells

Protein kinase C (PKC) plays a prominent role in immune signaling, and the paradigms for isoform selective signaling are beginning to be elucidated. Real-time microscopy was combined with molecular and biochemical approaches to demonstrate a role for PKC-ɛ in Fcγ receptor (FcγR)–dependent phagocytosis. RAW 264.7 macrophages were transfected with GFP-conjugated PKC isoforms, and GFP movement was followed during phagocytosis of fluorescent IgG–opsonized beads. PKC-ɛ, but not PKC-δ, concentrated around the beads. PKC-ɛ accumulation was transient; apparent as a “flash” on target ingestion. Similarly, endogenous PKC-ɛ was specifically recruited to the nascent phagosomes in a time-dependent manner. Overexpression of PKC-ɛ, but not PKC-α, PKC-δ, or PKC-γ enhanced bead uptake 1.8-fold. Additionally, the rate of phagocytosis in GFP PKC-ɛ expressors was twice that of cells expressing GFP PKC-δ. Expression of the regulatory domain (ɛRD) and the first variable region (ɛV1) of PKC-ɛ inhibited uptake, whereas the corresponding PKC-δ region had no effect. Actin polymerization was enhanced on expression of GFP PKC-ɛ and ɛRD, but decreased in cells expressing ɛV1, suggesting that the ɛRD and ɛV1 inhibition of phagocytosis is not due to effects on actin polymerization. These results demonstrate a role for PKC-ɛ in FcγR-mediated phagocytosis that is independent of its effects on actin assembly.

Effect of phagocytosis of erythrocytes and erythrocyte ghosts on macrophage phagocytic function and hydrogen peroxide production

Our previous studies have shown that an in vivo phagocytic challenge with IgG-coated erythrocytes can depress Kupffer cell complement and Fc receptor function, as well as decrease the survival rate following endotoxemia and bacteremia. In an effort to better understand the mechanism underlying these in vivo findings, the present study evaluated the in vitro effects of a phagocytic challenge with either IgG-coated erythrocytes (EIgG) or erythrocyte ghosts (GIgG) on macrophage phagocytic and respiratory burst activity. Elicited rat peritoneal macrophage (PM) monolayers were challenged with varying doses of EIgG, then the noninternalized EIgG were lysed hypotonically and the monolayers incubated for an additional hour prior to determining phagocytic function and PMA-stimulated hydrogen peroxide production. Challenge of PM with 1×106 EIgG per well had no effect, but challenge with 1×107 or 1×108 EIgG per well caused a dose-dependent depression of phagocytic function or hydrogen peroxide production. GIgG were formed by hypotonically lysing EIgG bound to PM at 4°C. The bound GIgG were phagocytized during a subsequent incubation at 37°C. Challenge with GIgG depressed phagocytic function only with the highest challenge dose tested (1×108 per well) and did not depress hydrogen peroxide production. The observation that prior phagocytic challenge with EIgG depressed macrophage function to a greater extent than challenge with GIgG supports our previous in vivo observations. Furthermore, these studies suggest that the internalization of erythrocyte contents, and not phagocytosis per se, plays an important role in determining macrophage host defense function.

Macrophage dysfunction following the phagocytosis of IgG-coated erythrocytes: production of lipid peroxidation products.

The phagocytosis of erythrocytes may contribute to the increased susceptibility to life‐threatening infections in patients with burn injury, sickle cell anemia, and malaria. The phagocytosis of Immunoglobulin G–coated erythrocytes (EIgG) is followed by a transient depression of several macrophage functions including phagocytosis, respiratory burst capacity, and killing of bacteria. The present study suggests the possibility that after erythrophagocytosis hemoglobin‐derived iron conspires with reactive oxygen products of the macrophage respiratory burst to cause oxidant damage to the phagocyte. Challenge of elicited peritoneal macrophages with EIgG phagocytosis was followed by an increase in lipid peroxidation as assessed by thiobarbituric acid–reactive substances (TBARS). Doses of EIgG associated with increased TBARS also caused a depression of Fc receptor–mediated phagocytosis and phorbol myristate acetate (PMA)–stimulated hydrogen peroxide production. Time course experiments demonstrated that the increase in TBARS coincided with the depression of macrophage function. There was no increase in TBARS following the phagocytosis of IgG‐coated erythrocyte ghosts, suggesting that hemoglobin iron is involved in the generation of TBARS. The phagocytosis of erythrocyte ghosts did not depress macrophage function. Since complement receptor–mediated phagocytosis does not stimulate the respiratory burst, the role of the respiratory burst in causing lipid peroxidation was assessed using the phagocytosis of complement‐coated erythrocytes. Phagocytic challenge with complement‐coated erythrocytes caused neither an increase in TBARS nor a depression of macrophage function. However, there was an increase in TBARS when the respiratory burst was stimulated with PMA following complement receptor–mediated phagocytosis of erythrocytes. These results suggest that hemoglobin iron and phagocyte‐generated oxidants collaborate to cause the depression of macrophage function following EIgG phagocytosis.

Long-term calibration considerations during subcutaneous microdialysis sampling in mobile rats.

The level at which implanted sensors and sampling devices maintain their calibration is an important research area. In this work, microdialysis probes with identical geometry and different membranes, polycarbonate/polyether (PC) or polyethersulfone (PES), were used with internal standards (Vitamin B(12) (MW 1355), antipyrine (MW 188) and 2-deoxyglucose (2-DG, MW 164)) and endogenous glucose to investigate changes in their long-term calibration after implantation into the subcutaneous space of Sprague-Dawley rats. Histological analysis confirmed an inflammatory response to the microdialysis probes and the presence of a collagen capsule. The membrane extraction efficiency (percentage delivered to the tissue space) for antipyrine and 2-DG was not altered throughout the implant lifetime for either PC- or PES membranes. Yet, Vitamin B(12) extraction efficiency and collected glucose concentrations decreased during the implant lifetime. Antipyrine was administered i.v. and its concentrations obtained in both PC- and PES-membrane probes were significantly reduced between the implant day and seven (PC) or 10 (PES) days post-implantation suggesting that solute supply is critical for in vivo extraction efficiency. For the low molecular weight solutes such as antipyrine and glucose, localized delivery is not affected by the foreign body reaction, but recovery is significantly reduced. For Vitamin B(12), a larger solute, the fibrotic capsule formed around the probe significantly restricts diffusion from the implanted microdialysis probes.

Role of PKC isoforms in the FcγR-mediated inhibition of LPS-stimulated IL-12 secretion by macrophages

Ligation of Fc receptors for immunoglobulin G (FcγRs) inhibits lipopolysaccharide (LPS)‐stimulated secretion of interleukin (IL)‐12 by macrophages. FcγR activation of protein kinase C (PKC) contributes to several functions of this receptor including phagocytosis, activation of the reduced nicotinamide adenine dinucleotide phosphate oxidase, and secretion of certain cytokines. Therefore, we tested the hypothesis that PKC mediates the FcγR inhibition of IL‐12 secretion by macrophages. In murine macrophages, FcγR ligation augmented LPS‐stimulated activation of PKC‐α and PKC‐δ but reduced IL‐12p40 secretion. Similarly, activation of PKC with phorbol 12‐myristate 13‐acetate (PMA) depressed LPS‐stimulated IL‐12p40 secretion, and depletion of PKC augmented LPS‐stimulated IL‐12p40 secretion. Antisense down‐regulation of PKC‐δ increased LPS‐stimulated IL‐12p40 secretion and fully prevented the effects of FcγR ligation or PMA on IL‐12p40 secretion. In contrast, down‐regulation of PKC‐ε blocked LPS‐stimulated secretion of IL‐12p40. Down‐regulation of PKC‐α had no effect on LPS‐stimulated IL‐12p40 secretion. The results suggest a negative role for PKC‐δ and a positive role for PKC‐ε in the regulation of LPS‐stimulated IL‐12p40 secretion.

Detection of in vivo Matrix Metalloproteinase Activity using Microdialysis Sampling and Liquid Chromatography-Mass Spectrometry

Matrix metalloproteinases (MMPs) are a family of endoproteases that break down extracellular matrix and whose upregulation contributes to several diseases. A liquid chromatography/tandem mass spectrometry (LC/MS/MS) method was developed to quantify MMP-1 and MMP-9 substrates and their N-terminal peptide products in samples obtained from implanted microdialysis sampling probes. In vitro studies with purified human MMP-1 and MMP-9 were used to optimize the assay and determine the effectiveness of the local delivery of a broad-spectrum MMP inhibitor, GM 6001. Localized delivery of GM 6001 at 10 microM was sufficient to completely inhibit product formation in vitro. In vivo studies in male Sprague-Dawley rats were performed with microdialysis probes implanted into the subcutaneous tissue. Directly after microdialysis probe implantation, infusions of the MMP-1 and MMP-9 substrates (50 microM each) resulted in recovered product concentrations of approximately 2 microM. During a 50 microM GM 6001 coinfusion with the substrates, a 30% and 25% reduction in product formation for the MMP-1 and MMP-9 substrates was obtained, respectively. Blank dialysates were negative for enzymatic activity that could cleave the MMP substrates. This method allowed for the activity of different MMPs surrounding the microdialysis probe to be observed during in vivo sampling.

Multiplexed cytokine detection of interstitial fluid collected from polymeric hollow tube implants--a feasibility study.

Cytokines are important cellular signaling proteins involved in inflammation, wound healing and are thought to direct the foreign body response to implanted materials. In this work, polyurethane tubes (25 mm length, 1.02 mm i.d., and 1.65 mm o.d.) were implanted into subcutaneous tissue of male Sprague-Dawley rats. The tubes served as the biomaterial and a means to collect the interstitial fluid that would be exchanged within the tube lumen and the surrounding tissue. After 3 and 7 days, the tubes were explanted and cytokines in the fluid were quantified with a multiplexed cytokine immunoassay. Six cytokines, interleukin-1beta (IL-1beta), IL-4, IL-6, IL-10, macrophage chemoattractant protein-1 (MCP-1), and tumor necrosis factor-alpha (TNF-alpha), were simultaneously quantified. All cytokine concentrations with the exception of IL-4 and TNF-alpha ranged between low pg/mL to mid ng/mL levels. Neither TNF-alpha nor IL-4 was detected from any sample. These results illustrate the potential of using the tube materials combined with bead-based immunoassays as a direct method for in vivo collection of multiple cytokines in low microliter sample volumes for fixed day biomaterial implant studies.

Modulation of the Foreign Body Reaction for Implants in the Subcutaneous Space: Microdialysis Probes as Localized Drug Delivery/Sampling Devices

Modulation of the foreign body reaction is considered to be an important step toward creation of implanted sensors with reliable long-term performance. In this work, microdialysis probes were implanted into the subcutaneous space of Sprague-Dawley rats. The probe performance was evaluated by comparing collected endogenous glucose concentrations with internal standard calibration (2-deoxyglucose, antipyrine, and vitamin B12). Probes were tested until failure, which for this work was defined as loss of fluid flow. In order to determine the effect of fbrous capsule formation on probe function, monocyte chemoattractant protein-1/CC chemokine ligand 2 (MCP-1/CCL2) was delivered locally via the probe to increase capsule thickness and dexamethasone 21-phosphate was delivered to reduce capsule thickness. Probes delivering MCP-1 had a capsule that was twice the thickness (500–600 μm) of control probes (200–225 μm) and typically failed 2 days earlier than control probes. Probes delivering dexamethasone 21-phosphate had more fragile capsules and the probes typically failed 2 days later than controls. Unexpectedly, extraction efficiency and collected glucose concentrations exhibited minor differences between groups. This is an interesting result in that the foreign body capsule formation was related to the duration of probe function but did not consistently relate to probe calibration.