Evidence for Paracrine Signaling Between Macrophages and Bovine Adrenal Chromaffin Cell Ca2+ Channels

Kevin P. M. Currie, Zhong Zhou, and Aaron P. Fox

Department of Pharmacological and Physiological Sciences, The University of Chicago, Chicago, Illinois 60637


    ABSTRACT
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

Currie, Kevin P. M., Zhong Zhou, and Aaron P. Fox. Evidence for Paracrine Signaling Between Macrophages and Bovine Adrenal Chromaffin Cell Ca2+ Channels. J. Neurophysiol. 83: 280-287, 2000. The adrenal gland contains resident macrophages, some of which lie adjacent to the catecholamine producing chromaffin cells. Because macrophages release a variety of secretory products, it is possible that paracrine signaling between these two cell types exists. Of particular interest is the potential paracrine modulation of voltage-gated calcium channels (ICa), which are the main calcium influx pathway triggering catecholamine release from chromaffin cells. We report that prostaglandin E2 (PGE2), one of the main signals produced by macrophages, inhibited ICa in cultured bovine adrenal chromaffin cells. The inhibition is rapid, robust, and voltage dependent; the activation kinetics are slowed and inhibition is largely reversed by a large depolarizing prepulse, suggesting that the inhibition is mediated by a direct G-protein beta gamma subunit interaction with the calcium channels. About half of the response to PGE2 was sensitive to pertussis toxin (PTX) incubation, suggesting both PTX-sensitive and -insensitive G proteins were involved. We show that activation of macrophages by endotoxin rapidly (within minutes) releases a signal that inhibits ICa in chromaffin cells. The inhibition is voltage dependent and partially PTX sensitive. PGE2 is not responsible for this inhibition as blocking cyclooxygenase with ibuprofen did not prevent the production of the inhibitory signal by the macrophages. Nor did blocking the lipoxygenase pathway with nordihydroguaiaretic acid alter production of the inhibitory signal. Our results suggest that macrophages may modulate ICa and catecholamine secretion by releasing PGE2 and other chemical signal(s).


    INTRODUCTION
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

Interactions between the immune system and the adrenal gland are well documented. For instance, there is elevated catecholamine release by the adrenal medulla during periods of stress and infection due both to increased sympathetic stimulation and peripheral mechanisms (Zhou and Jones 1993). In addition, cytokines stimulate the hypothalamic-pituitary-adrenal axis leading to increased glucocorticoid production by the adrenal cortex; this results in a negative feedback immunosuppressive effect (Buckingham et al. 1996; Turnbull and Rivier 1995). As opposed to these indirect neurogenic effects of the immune system on adrenal function, it is becoming increasingly clear that there are direct paracrine interactions within the adrenal gland itself (Marx et al. 1998; Nussdorfer and Mazzocchi 1998). Interest in this field has focused largely on the actions of intra adrenal cytokines, especially on cortical cells. These cytokines are produced both by adrenal cells themselves and by macrophages. Both rat and human adrenal glands have a population of resident macrophages distributed throughout the cortex and medulla (Gonzalez-Hernandez et al. 1994; Schober et al. 1998), some of which lie adjacent to the catecholamine producing chromaffin cells. This makes them ideally situated to participate in paracrine signaling to the chromaffin cells and potentially modulate catecholamine release. Such signaling has been suggested previously as a subpopulation of the resident macrophages contain neurotrophin-4 (NT4) and chromaffin cells express the Trk A receptor that binds NT4 (Schober et al. 1998). Other studies have demonstrated that a peptide released by human monocytes stimulated catecholamine release from cultured adrenal chromaffin cells (Jones et al. 1993; Roberts et al. 1996).

Of particular interest is the question of whether macrophages release substances that alter calcium signals in chromaffin cells, especially by modulating voltage-gated calcium channels (ICa) that are the main calcium influx pathway triggering secretion (Boarder et al. 1987). Immune-system signals modulate ICa in other types of cells; ICa is enhanced by interleukin-1beta in vascular smooth muscle cells (Wilkinson et al. 1996). In contrast, interleukin 1beta (Plata-Salaman and ffrench-Mullen 1992) and thromboxane A2 agonists (Hsu et al. 1996) both inhibit ICa in hippocampal neurons, whereas prostaglandin E2 (PGE2) inhibits ICa in sympathetic ganglion neurons (Ikeda 1992).

PGE2, one of the main metabolites released by activated macrophages, has been shown to specifically bind to adrenal chromaffin cells, release intracellular calcium stores, and stimulate calcium influx through voltage-independent channels and modulate catecholamine release (Ito et al. 1991; Karaplis et al. 1989; Marley et al. 1988; Mochizuki-Oda et al. 1991; Yokohama et al. 1988). Prostaglandins are produced by cyclooxygenase (COX), which catalyzes the first two steps in their synthesis from arachidonic acid (Vane et al. 1998). Two different isoforms of the enzyme have been identified: COX-1, which is constitutively active, and COX-2, which is inducible. Typically, substantially elevated PGE2 levels are only observed several hours after activation of macrophages due to induction of COX-2 activity (Lee et al. 1992; O'Sullivan et al. 1992; Pueringer and Hunninghake 1992). However, rapid production of PGE2 through the constitutively active COX-1 pathway is possible as demonstrated by application of exogenous arachidonic acid to cultured macrophage cell lines (Stenson et al. 1981).

These observations raised the possibility that macrophages could inhibit ICa in adrenal chromaffin cells and thereby modulate catecholamine release. Moreover, this signaling could occur on both a slow and more rapid time scale: the slow pathway mediated by the well-documented induction of synthetic enzymes (such as COX-2) in the macrophages during periods of immune-system activation and the rapid pathway by production of PGE2 or other arachidonic acid metabolites by the constitutively active COX-1, which may play a role under both pathophysiological conditions and normal physiological functioning of the gland. To address the latter possibility, we chose to investigate potential signaling between macrophages and chromaffin cells in two ways. First, as PGE2 is an attractive candidate for this kind of signaling, we investigated the effects of exogenously applied PGE2 on ICa in chromaffin cells. The second approach involved activating a macrophage cell line with lipopolysaccharide (endotoxin) to determine whether these cells rapidly secrete signaling molecules that could alter ICa activity. Our results demonstrate inhibition of ICa in adrenal chromaffin cells by PGE2 and an unidentified signaling molecule(s) that is released rapidly from macrophages.


    METHODS
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

Cell culture

Chromaffin cells were prepared by digestion of bovine adrenal glands with collagenase and purified by density gradient centrifugation as previously described (Artalejo et al. 1992). The cells were plated on collagen-coated glass coverslips in 35-mm tissue culture dishes (2 ml of cell suspension; 0.15-0.2 × 106 cells/ml) and maintained in an incubator at 37°C in an atmosphere of 93% air and 7% CO2 with a relative humidity of 90%. Fibroblasts were suppressed effectively with cytosine-arabinoside (10 µM), leaving relatively pure chromaffin cell cultures. Although mixed, the cultures were somewhat enriched for epinephrine containing over norepinephrine-containing cells. Half of the incubation medium was exchanged every day. This medium consisted of DMEM/F12 (1:1) (Gibco) supplemented with fetal bovine serum (10%), glutamine (2 mM), penicillin/streptomycin (100 unit/ml and 100 µg/ml), cytosine arabinoside (10 µM), and 5-fluorodeoxyuridine (10 µM).

J774.1 cells, a murine macrophage cell line (Snyderman at al. 1977), were grown in tissue culture flasks or dishes. Cells were maintained at 37°C in humidified air containing 5% CO2 in RPMI culture medium (Gibco) supplemented with 5% fetal bovine serum and penicillin/streptomycin (100 unit/ ml and 100 µg/ml). Cells were passaged approximately once a week by mechanical trituration.

Electrophysiology

Chromaffin cells were voltage clamped in the whole cell configuration of the patch-clamp technique (Hamill et al. 1981) using an Axopatch 1C amplifier (Axon Instruments) at a holding potential of -80 mV, and ICa were activated by step depolarizations. Leak currents were generated by averaging 16 hyperpolarizing sweeps (steps or ramps). All the data reported in this paper were capacitance and leak subtracted. The data were filtered at 2 kHz and then digitized at 100 µs/point. Series resistance was compensated partially (approx 80%) using the series resistance compensation circuit of the Axopatch-1C amplifier. Electrodes were pulled from microhematocrit capillary tubes (Drummond) and coated with silicone elastomer (Sylgard; Dow Corning). After fire polishing, final electrode resistances when filled with the CsCl-based patch pipette solution (see following section) were ~1.5-3.0 MOmega . Voltage protocols and data analysis were carried out in AxoBasic. Data are reported as means ± SE, and statistical significance was determined using paired or independent Student's t-test. All recording was performed at room temperature (~23°C).

Solutions

Electrodes were filled with (in mM) 110 CsCl, 4 MgCl2, 20 HEPES, 10 EGTA, 0.35 GTP, 4 ATP, and 14 creatine phosphate, pH = 7.3 (adjusted by CsOH) and osmolarity was ~310 mOsm. The NaCl-based extracellular recording medium contained (in mM) 140 NaCl, 2 KCl, 10 glucose, 10 HEPES, and 10 CaCl2as well as 0.3-1.0 µM tetrodotoxin (TTX), pH = 7.3 (adjusted with NaOH), and the osmolarity was ~310 mOsm. In a few experiments, the TTX was omitted. Nisoldipine was prepared as a stock solution (10 mM) in ethanol and stored, protected from light at -20°C. It was added to all extracellular solutions (1 µM) to block any facilitation ICa (L-type) present.

PGE2 (Calbiochem) was prepared as a stock solution in DMSO and aliquots frozen. Final dilutions yielded DMSO concentrations of <0.03%, which had no effect on the currents by itself. Interleukin-1beta and interleukin-6 (Sigma) were prepared as stocks of 10 µg/ml and aliquots frozen until use. Ibuprofen and nordihydroguaiaretic acid (NDGA; Sigma) were both prepared fresh by dilution in H2O with the addition of NaOH. Final dilution yielded no alteration on the pH of the recording or incubation medium. Lipopolysaccharide (LPS) from Escherichia coli (serotype 055:B5; Sigma) was dissolved in sterile H2O (25 mg/ml) and aliquots stored at 4°C for 2-4 wk.

Application of solutions and preparation of conditioned medium

The recording bath was <1 cm in diameter with a volume of around 250-350 µl. The bath solution was gravity fed from reservoirs at a flow rate of 3-4 ml per minute, that ensured efficient perfusion of the recording chamber. Agonists and antagonists were applied to the cells by including them in the recording solution and washing them into the bath. There was a latency between switching solutions at the reservoirs and the drugs reaching the cell due to "dead space" in the tubing leading to the bath. This accounted for most of the delay seen between agonist application and inhibition of ICa. omega -conotoxin GVIA (35-50 µl) was added directly to the bath, with the flow of extracellular solution stopped, at 10 times the desired final concentration. Thus omega -conotoxin GVIA was added at 10 µM to give a final concentration of ~1 µM.

For experiments in which J774 cells were present in the recording chamber along with the chromaffin cells, the J774 cells were grown on tissue culture dishes until almost confluent. Cells then were removed from the dish by gentle trituration and resuspended at a density of roughly 0.2-1 × 106 in NaCl-based recording medium. Once a chromaffin cell had been voltage clamped, the flow of solution through the recording chamber was stopped, and 50-75 µl of the cell suspension was added directly to the bath. The J774 cells quickly settled and adhered to the coverslip. After several minutes the flow of solution through the bath was resumed to wash away any cells that had not stuck to the coverslip.

For experiments with conditioned media the J774 cells were grown on 10 cm diameter tissue culture dishes until confluent. Cells then were washed several times with NaCl-based recording medium and then incubated for 5-10 min with 7-8 ml of either NaCl-based recording medium (control conditioned media) or NaCl-based recording medium containing 100-250 µg/ml LPS (LPS-conditioned media). The conditioned media was collected and applied directly to chromaffin cells by washing through the bath using the gravity fed perfusion system. TTX and nisoldipine were omitted from the NaCl recording medium before conditioning and were added after the solution was removed from the J774 cells.


    RESULTS
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

PGE2 inhibits ICa in chromaffin cells in a voltage-dependent manner

PGE2, an important metabolite produced by activated macrophages, was applied to the chromaffin cells by continuous perfusion through the recording chamber. In virtually every cell tested, PGE2 produced a rapid, reversible inhibition of ICa similar to the response illustrated in Fig. 1. Figure 1A plots peak-current amplitude as a function of time. This cell was depolarized every 10 s to +20 mV from HP = -80 mV. Approximately 70% of the current was inhibited when PGE2 (300 nM) was applied. Figure 1B shows representative currents obtained during this experiment; the currents were inhibited and activation was slowed. In other experiments, PGE2 was applied at concentrations of 1 nM to 1 µM, and the resulting dose-response curve (not shown) yielded an EC50 for the inhibition of ICa of ~10 nM. Application of a supramaximal dose of PGE2 (300 nM), similar to that shown in Fig. 1, produced a mean inhibition of 53 ± 4.5% (n = 20). Multiple applications of PGE2 to the same cell produced repeated inhibition of ICa, suggesting there was little desensitization of the response; however, the washout was often slow and incomplete.



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Fig. 1. Prostaglandin E2 (PGE2) inhibits ICa in adrenal chromaffin cells. A: peak current amplitude is plotted against time. Depolarizations from HP = -80 to +20 mV, lasting 20 ms, were applied every 10 s. PGE2 (300 nM) was applied to the cell by continuous perfusion through the recording chamber as indicated by the horizontal bar. B: current records taken from the same cell as shown in A before (control), during (PGE2), and after washout (wash) of PGE2.

Activation of G-protein-coupled receptors by various transmitters/hormones can inhibit ICa by multiple pathways (Hille 1994) one of which is thought to involve direct binding of the G-protein beta gamma subunits to the channel (Herlitze et al. 1996; Ikeda 1996; for review, see Dolphin 1998). This type of inhibition is voltage dependent and characterized by slowed activation kinetics of ICa, similar to that shown in Fig. 1B, and relief of the inhibition by a conditioning prepulse (Bean 1989; Elmslie et al. 1990; Penington et al. 1991). Figure 2A shows an experiment where a conditioning prepulse (50-ms duration to +100 mV, applied 10 ms before the activation of ICa) relieved ~65% of the inhibition produced by PGE2. On average these prepulses relieved 59 ± 3% (n = 13) of the inhibition and reversed the kinetic slowing (Fig. 2B). Please note that 1 µM nisoldipine was present throughout these experiments, so the prepulse increases in ICa were due to relief of inhibition not recruitment of L-type channels.



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Fig. 2. PGE2 inhibition of ICa is relieved by a conditioning prepulse. A: plots of 3 superimposed current records. Current trace labeled "control" was obtained by depolarizing this cell from HP = -80 to +20 mV for 20 ms, in the absence of PGE2, whereas the current trace labeled "PGE2" was obtained with an identical test pulse in the presence of 300 nM PGE2. The third current trace, labeled "PGE2 & prepulse," was obtained in the presence of PGE2, but 10 ms before the test pulse there was a 50-ms prepulse to +100 mV; the prepulse reversed the kinetic slowing and partially relieved the current inhibition. B: plots of average peak current amplitude for the 3 different experimental conditions from 13 cells like that shown in A. All peak current amplitudes were normalized to control (before application of PGE2).

PGE2 inhibition is mediated by both PTX-sensitive and -insensitive G proteins

Most examples of voltage-dependent inhibition of ICa are mediated by the Gi/Go family of G proteins and can be blocked by pertussis toxin (PTX), which disrupts the coupling of these G proteins with their receptors. We therefore tested the PTX sensitivity of the PGE2 inhibition. Cells were preincubated for 18-24 h with 300 ng/ml PTX and then PGE2 applied as before. In control cells (cells from the same cultures and recorded from on the same days as the treated cells), 300 nM PGE2 inhibited ICa by 47 ± 3.3% (n = 11), whereas in PTX-treated cells the inhibition was 21 ± 1.5% (n = 13; P < 1 × 10-6; Fig. 3). The PTX-insensitive inhibition still exhibited the voltage-dependent characteristics of slowed activation kinetics and prepulse reversal. Our results suggest that both PTX-sensitive and -insensitive G proteins are involved in the PGE2 response.



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Fig. 3. PGE2 inhibition of ICa is mediated by both pertussis-toxin (PTX)-sensitive and -insensitive G proteins. Average inhibition of ICa produced by 300 nM PGE2 is shown for 11 control cells and 13 cells pretreated for 18-24 h with 300 ng/ml PTX. Control cells were from the same cultures and were recorded from on the same days as PTX-treated cells. Inhibition produced in PTX-treated cells was significantly reduced compared with control cells (P < 1 × 10-6).

Both N- and P/Q-type ICa were inhibited by PGE2

Under our recording conditions (with L-type channels blocked by nisoldipine), we have shown previously that ICa consists of ~50% N-type and ~50% P/Q-type channels (Currie and Fox 1996, 1997). Furthermore both these current components are inhibited by activation of P2Y purinergic receptors. Consistent with these data, application of 1-2 µM omega -conotoxin GVIA to selectively block the N-type ICa reduced the current amplitude by 49 ± 3.1% (n = 4). PGE2 (300 nM) inhibited the remaining P/Q-type current by 33 ± 3.7% (n = 4). Our results suggest that both N- and P/Q-type current components were inhibited by PGE2 and that the N-type current was inhibited to a greater extent than the P/Q type current.

J774 macrophages rapidly release an inhibitor of ICa when stimulated with lipopolysaccharide (endotoxin)

From the preceding results it was clear that PGE2 inhibited ICa in adrenal chromaffin cells. Typically there is a delay of several hours after macrophage activation (due to induction of COX-2 activity) before there is a substantial elevation of PGE2 production and release. Macrophages also express the constitutively active form of the enzyme (COX-1) and so have the ability to rapidly produce PGE2 from arachidonic acid. To determine whether macrophages could rapidly produce PGE2 or other paracrine modulators of ICa on exposure to endotoxin (a lipopolysaccharide component of bacterial cell walls commonly used to activate macrophages), a mouse macrophage cell line (J774.1) was exposed to short applications (1-10 min) of high concentrations (100-250 µg/ml) of lipopolysaccharide (LPS) from Escherichia coli; chemical signals secreted by the macrophages were tested on chromaffin cell ICa.

Two approaches were used: addition of J774 cells directly to the recording chamber containing chromaffin cells followed by exposure to LPS and generation of conditioned medium from culture dishes of J774 cells and application of this directly to the chromaffin cells. The first approach is illustrated in Fig. 4. After establishing whole cell recording from a chromaffin cell, the flow of solution through the recording chamber was stopped and a suspension of J774 cells added to the chamber. The cells settled with minutes and flow of the bath was resumed. LPS then was applied to the cells, and the flow of solution through the bath was again stopped to facilitate the accumulation of any substances released from the macrophages. This produced a very rapid inhibition of ICa amplitude and a slowing of the activation kinetics (Fig. 4B). The inhibition was washed out rapidly once flow of solution through the bath was resumed. In 31 cells, the mean inhibition produced in the manner just described was 37 ± 1.2%. Application of LPS to the chromaffin cells in the absence of J774 cells had no effect on ICa (Fig. 5). Similarly when J774 cells were applied to the bath as described in the preceding text and the flow of solution stopped, there was no inhibition of ICa unless LPS was also present (Fig. 5). Prior incubation of the chromaffin cells with PTX reduced the inhibition produced by the same protocol to 12 ± 5% (n = 5).



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Fig. 4. Exposure of the J774 macrophage cell line to lipopolysaccharide rapidly inhibits ICa recorded from nearby chromaffin cells. A: J774 cells were added to the recording chamber and settled onto the coverslip adjacent to the chromaffin cells within minutes. Lipopolysaccharide (LPS; 100-250 µg/ml) was washed rapidly into the chamber and the flow of solution stopped (indicated by bar) to allow accumulation of any chemical signals released from the J774 cells. Graph plots peak current amplitude vs. time and shows that LPS stimulation led to a rapid inhibition of ICa. This inhibition was rapidly washed out when fresh solution was perfused through the chamber. B: current records from the cell shown in A. Currents were recorded before application of LPS (control), in the presence of LPS with the flow of solution through the bath stopped (LPS), and after washout of LPS with the bath solution continuously flowing (wash).



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Fig. 5. LPS does not directly inhibit ICa in the chromaffin cells but requires the presence of J774 macrophage cells. Calcium current amplitudes recorded from chromaffin cells were normalized to controls recorded in the absence of either J774 cells or LPS in the recording chamber. Application of LPS to chromaffin cells alone (LPS) did not have a significant effect on ICa. Presence of J774 cells in the recording chamber was also without effect. Application of LPS to the recording chamber with J774 cells present (J774/LPS) produced a significant inhibition of chromaffin cell ICa (P < 0.05).

Application of conditioned medium collected from J774 cells to chromaffin cells produced similar results (Fig. 6). J774 cells were incubated for 5-10 min with either NaCl-based recording medium to produce control conditioned medium or NaCl-based recording medium containing LPS (100-250 µg/ml) to produce LPS-conditioned medium. This conditioned medium then was collected and applied directly to the chromaffin cells by continuous perfusion through the recording chamber. Control conditioned medium had little or no effect on ICa, but LPS-conditioned medium reversibly inhibited ICa (Fig. 6, A and B). The inhibition slowed the activation kinetics of ICa and was relieved by a depolarizing prepulse (Fig. 6B). Normalizing the data to the control conditioned media showed that LPS-conditioned media inhibited ICa by 18 ± 2.3% (n = 12; P < 0.001) and a prepulse to +100 mV reduced this inhibition to 3 ± 2% (n = 8; Fig. 7). These data are consistent with the idea that a chemical signal was rapidly (within seconds to minutes) released from the J774 cells and acted on a G-protein-coupled receptor to inhibit ICa in the chromaffin cells.



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Fig. 6. Conditioned medium collected from J774 cells inhibits ICa in chromaffin cells. J774 cells were incubated for 5-10 min in NaCl-based recording medium in the presence (LPS-conditioned media) or absence (control conditioned media) of LPS. This conditioned medium then was removed from the J774 cells and applied to chromaffin cells by continuous perfusion through the recording chamber. A: peak ICa amplitude is plotted against time using depolarizations identical to those described in Fig. 1. Cells were perfused continuously with NaCl-based recording medium (wash), control conditioned medium (ctl cm), or LPS-conditioned medium (LPS cm). Control conditioned medium had little effect on ICa, whereas LPS-conditioned medium produced a reversible inhibition of ICa. Current labeled "p" was preceded by a prepulse to +100 mV (see B). B: plots of 4 superimposed current records from the cell shown in A. Currents were recorded before application of conditioned medium (wash), in the presence of control conditioned medium (control "cm"), in the presence of LPS-conditioned medium (lps cm), and in the presence of LPS-conditioned medium but preceded by a prepulse to +100 mV (lps cm & prepulse).



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Fig. 7. LPS-conditioned medium produces a voltage-dependent inhibition of ICa. Mean data for cells that responded to conditioned media (like the example shown in Fig. 6) is plotted (nonresponding cells have been excluded). Current amplitudes were normalized relative to control conditioned medium (control) recorded before application of LPS conditioned medium (LPS). Shown are the average normalized peak current amplitudes recorded in the presence of control conditioned medium, LPS-conditioned medium, and LPS-conditioned medium but preceded by a 50-ms prepulse to +100 mV. (*, statistical significance from control conditioned medium; P < 0.05.) (Pooled data from cells that responded showed that the average effect of control conditioned medium was a slight reduction of 4 ± 0.7%, n = 12.)

Please note that the percentage of cells responding was variable from week to week for both the conditioned media experiments and the experiments in which the J774 cells were present in the recording chamber. The reasons for this are not clear but are considered in the discussion.

Rapidly released inhibitor is not PGE2

To determine if the transmitter released by the J774 cells was PGE2, the experiments shown in Figs. 6 and 7 were repeated with conditioned medium from J774 cells that had been pretreated for 1-2 h with 30-100 µM ibuprofen, which blocks activity of both COX-1 and COX-2. Control dishes of J774 cells were treated in the same manner except with the omission of ibuprofen. Ibuprofen also was present during exposure to LPS and so was present in all conditioned medium applied to the chromaffin cells. Ibuprofen itself had no direct action on ICa. The amplitude of ICa was 1,403 ± 268 pA before application and 1,359 ± 264 pA (n = 5) during application of control conditioned medium (no LPS) containing 30 µM ibuprofen.

Chromaffin cells first were exposed to LPS-conditioned medium from untreated J774 cells. After obtaining a response the cell then was washed and LPS-conditioned medium from ibuprofen-treated J774 cells was applied (Fig. 8A). There was no difference in the inhibition of ICa produced by conditioned media from control cells (17 ± 4%, n = 4) or ibuprofen-treated cells (18 ± 3%, n = 4; Fig. 8B).



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Fig. 8. Block of cyclooxygenase or lipoxygenase enzymes in J774 cells does not prevent the inhibition of ICa produced by LPS conditioned medium. A: peak ICa amplitude is plotted against time, using depolarizations identical to those described in Fig. 1. Bars indicate application by continuous perfusion of LPS-conditioned medium collected from control J774 cells (control) or J774 cells that had been pretreated with ibuprofen, which blocks cyclooxygenase enzymes (labeled "ibuprofen"). Ibuprofen treatment did not prevent the inhibition. B: average data from 4 cells like that shown in A. There was no significant difference in the mean percentage inhibition of ICa produced by LPS conditioned medium collected from control or ibuprofen-treated J774 cells. C: peak ICa amplitude is plotted against time; the bars indicate application of LPS-conditioned medium from control J774 cells and J774 cells treated with nordihydroguaiaretic acid (NDGA), which blocks lipoxygenase enzymes. D: average data from 4 cells like that in C. There was no significant difference in the inhibition produced by LPS-conditioned medium collected from control or NDGA-treated J774 cells.

Arachidonic acid also can be metabolized by lipoxygenase enzymes to produce signaling molecules such as leukotrienes or other active metabolites. The activity of both 12-lipoxygenase and 5-lipoxygenase can be blocked by nordihydroguaiaretic acid (NDGA). To determine if a product of this pathway was responsible for the rapidly produced inhibition, experiments were performed as described in the preceding text except J774 cells were incubated for ~2 h with 30 µM NDGA. There was no difference in the mean inhibition of ICa produced by conditioned medium from control (17.5 ± 2.1%; n = 4) or NDGA-treated (19 ± 1.9%; n = 4) J774 cells (Fig. 8, C and D), suggesting that these pathways are not involved in the response.

It seemed unlikely that cytokines were responsible for the rapid signaling observed because it takes hours rather than minutes after activation of macrophages before there is an up-regulation of cytokine synthesis (Lin et al. 1994; Yoo et al. 1995). However, interleukins (including IL-1 and IL-6) have been detected in the adrenal gland (Nussdorfer and Mazzocchi 1998), and IL-1beta is known to inhibit ICa in hippocampal neurons (Plata-Salaman and ffrench-Mullen 1992). Therefore IL-1beta and IL-6 (30-100 ng/ml) were tested to determine whether they produced an inhibition of ICa in the chromaffin cells. Chromaffin cells were exposed to the interleukins by continuous perfusion through the recording chamber. Neither interleukin had an effect on ICa. In four cells tested, the amplitude of ICa was 1,126 ± 119 pA under control conditions and 1,101 ± 101 pA in the presence of IL-1beta . Similarly, in five different cells, the control ICa amplitude was 995 ± 32 pA and in the presence of IL-6 was 984 ± 33 pA.


    DISCUSSION
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

It is known that resident macrophages within the adrenal gland lie next to chromaffin cells. Macrophages are secretory cells that synthesize over 100 distinct products (Nathan 1987). The aim of this study was to investigate possible paracrine signaling between macrophages and adrenal chromaffin cells. In particular, these studies were focused on modulation of ICa as these channels are important targets for regulation of catecholamine release. PGE2, synthesized from arachidonic acid by the cyclooxygenase pathway, is one of the primary products secreted by macrophages. PGE2 is known to have actions on adrenal chromaffin cells including elevation of intracellular calcium and modulation of catecholamine release (Ito et al. 1991; Karaplis et al. 1989; Mochizuki-Oda et al. 1991; Yokohama et al. 1988) but its actions on ICa were unknown. However, PGE2 does inhibit ICa in sympathetic ganglion neurons (Ikeda 1992), which are ontogenetically similar to chromaffin cells. It therefore seemed to be an attractive candidate to participate in paracrine inhibition of ICa in chromaffin cells.

Our results demonstrate that PGE2 produced a robust inhibition of both N- and P/Q-type ICa in virtually every cell tested. In addition to the reduction in current amplitude, the activation kinetics of ICa were slowed. Conditioning prepulses reversed the slowing of activation and partially relieved the reduction in current amplitude. These features are characteristic of G-protein-mediated inhibition of N- and P/Q-type ICa in chromaffin cells, neurons, and other cell types (Bean 1989; Currie and Fox 1996; Dolphin 1995; Elmslie et al. 1990; Hille 1994; Penington et al. 1991). Most examples of this type of inhibition are mediated by PTX-sensitive G proteins. In the chromaffin cells, it appears that PGE2 couples to both PTX-sensitive and -insensitive G proteins to inhibit ICa similar to results obtained in sympathetic neurons (Ikeda 1992). It is not clear whether the same PGE2 receptor couples to multiple G proteins or whether there are multiple receptors activated concomitantly. There are very few readily available, selective pharmacological agents for prostanoid receptors so the subtype(s) involved in this response were not characterized.

Macrophages express the constitutive (COX-1) as well as the inducible (COX-2) form of cyclooxygenase. Substantially elevated production of PGE2 is usually not seen until several hours after activation of macrophages in part due to induction of COX-2 (Lee et al. 1992; O'Sullivan et al. 1992; Pueringer and Hunninghake 1992). However, it is possible for PGE2 to be produced rapidly by COX-1 as demonstrated by addition of exogenous arachidonic acid to macrophage cell lines (Stenson et al. 1981). This raised the possibility that macrophages could signal rapidly to chromaffin cells through the production of PGE2 (or other arachidonic acid metabolite) in addition to slower signaling through induction of synthetic enzymes such as COX-2 or synthesis of cytokines and related products.

This rapid signaling pathway was investigated using a mouse macrophage cell line (J774.1) stimulated with high concentrations of lipopolysaccharide (LPS), commonly used to activate macrophages. Addition of J774 cells to a recording chamber containing chromaffin cells had no effect on ICa unless LPS also was added to the bath. With LPS and J774 cells present, and the flow of solution through the bath stopped to allow accumulation of any released chemical signals, ICa amplitude was inhibited rapidly and activation kinetics were slowed; prepulses relieved a portion of the inhibition. PTX pretreatment of chromaffin cells significantly reduced the inhibition. Thus these results suggest the existence of rapid signaling between the immune system and chromaffin cells.

To confirm these observations, conditioned medium was collected from J774 cells and applied directly to chromaffin cells. This produced a comparable inhibition of ICa. Interestingly, responses both to LPS-activated macrophages present alongside chromaffin cells and to conditioned media were much less consistent than direct application of PGE2. The proportion of cells responding to conditioned medium was low and variable from week to week. The reasons for this variability are uncertain but may include: only a subpopulation of the chromaffin cells respond to the macrophage-derived chemical mediator; variability in J774 cell density and/or properties; variability between batches of LPS; and variable amounts of the macrophage-derived mediator produced and/or potential degradation or decay of this chemical once released. Nonetheless despite the problems arising from this variability, responses from many cells were obtained allowing the inhibition to be characterized.

The rapid inhibition produced by the LPS-conditioned medium was not mediated by PGE2 as blocking COX activity in the J774 cells using ibuprofen did not suppress the inhibition. The lipoxygenase pathway, which metabolizes arachidonic acid into leukotrienes, did not appear to be involved in the rapid signaling, as blockade of this pathway did not prevent inhibition of ICa by the LPS-conditioned medium. It is possible that there are multiple signaling molecules involved in the response and at present the identity of the inhibitor(s) remains unknown. The observation that the macrophage-derived inhibitor was stable in LPS-conditioned medium suggests it is not nitric oxide, which has a short half life once released from macrophages. The same is true for the epoxyeicosatrienoic acids, which are short-lived arachidonic acid metabolites produced by the cytochrome P450 pathway (Imig 1999). Activation of macrophages stimulates the synthesis of cytokines so it appears unlikely that large amounts could be released rapidly enough to account for the inhibition reported in this paper. However, IL-1 and IL-6 have been reported in the adrenal gland (Nussdorfer and Mazzocchi 1998) and IL-1beta inhibits ICa in hippocampal neurons (Plata-Salaman and ffrench-Mullen 1992). Both IL-1beta and IL-6 were applied directly to chromaffin cells but neither was found to have an effect on ICa.

Paracrine signaling within the adrenal gland is likely to be a complex web of interactions. Chromaffin cells are known to influence the functioning of adrenocortical cells; these cells send signals back to the chromaffin cells as well (Nussdorfer 1996; Pignatelli et al. 1998). It is also becoming apparent that resident macrophages can participate in paracrine signaling within the adrenal gland; locally produced cytokines act on the adrenal cortex to stimulate steroid production (Marx et al. 1998; Nussdorfer and Mazzocchi 1998). Conversely, glucocorticoids are known to suppress cytokine production and induction of COX-2 (Buckingham et al. 1996; Turnbull and Rivier 1995), which contributes to the negative feedback immunosuppressive actions of the hypothalamic-pituitary-adrenal axis. There are fewer reports of paracrine interactions between macrophages and chromaffin cells, but these also may operate bidirectionally; catecholamines are known regulators of immune-system function (Coffey and Hadden 1985; Johnson et al. 1981) and histogranin, a recently described peptide released from chromaffin cells, has been shown to stimulate immune cells including macrophages (Lemaire et al. 1995). Conversely, stimulation of catecholamine secretion by a peptide released from monocytes (Jones et al. 1993; Roberts et al. 1996) and the induction of c-fos immunoreactivity in chromaffin cells by NT4, which is contained in a subpopulation of the resident macrophage cells (Schober et al. 1998) have been reported. PGE2 also has been reported to augment or directly stimulate catecholamine release possibly by stimulating extracellular calcium influx through voltage-independent channels (Karaplis et al. 1989; Marley et al. 1988; Yokohama et al. 1988). However, concentrations of PGE2 in the nanomolar range suppress catecholamine release stimulated by nicotine (Karaplis et al. 1989). The results reported in this paper suggest that PGE2 also may suppress catecholamine release by reducing calcium influx through ICa. Further study will be required to elucidate the precise balance between these two seemingly opposing mechanism on catecholamine release.

In summary, this paper identifies a novel paracrine signaling pathway between macrophages and adrenal chromaffin cells that may regulate catecholamine release through modulation of ICa. It is possible that during periods of immune-system activation (infection), induction of COX-2 may elevate locally produced PGE2, suppress calcium influx into the chromaffin cells, and oppose the increased sympathetic stimulation to help prevent excessive elevations in circulating catecholamines. The rapid production of a paracrine inhibitor(s) of ICa also lends a further dimension to macrophage-chromaffin cell interactions. It may facilitate a more dynamic signaling pathway that potentially could play a role in the normal physiological functioning of the adrenal medulla as well as during periods of immune-system activation.


    FOOTNOTES

Address for reprint requests: K. Currie, The University of Chicago, Dept. of Pharmacological and Physiological Sciences, 947 E. 58th St., Chicago, IL 60637.

The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

Received 13 July 1999; accepted in final form 15 September 1999.


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ABSTRACT
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