Local Anesthetic Effects on Priming and Activation of Human Neutrophils

The study protocol was approved by the University of Virginia Institutional Review Board.

Human venous blood was obtained from healthy donors who had not used any medication for at least 2 weeks. Blood was heparinized (10 U/ml), and hPMNs were isolated by a one-step separation procedure. After 40 min of centrifugation at 500 g , hPMNs were washed three times with Hank’s balanced salt solution (HBSS; containing 10 U/ml heparin) and centrifuged after each washing step at 350 g  (20 min after first wash, 10 min after second and third wash). hPMNs were then resuspended in 5 ml HBSS and counted using a hemocytometer. Unopette in vitro  Diagnostic System (Becton Dickinson, Franklin Lakes, NJ) was used for the enumeration of hPMNs in the suspension, which was then diluted with HBSS to obtain a final neutrophil suspension of 5 × 10⁶cells/ml. Purity of our hPMN suspension, assessed by morphology, exceeded 98%. The viability of neutrophils was checked by Trypan blue exclusion and was always found to be greater than 94%. All preparation and assays were performed at room temperature.

We used the cytochrome-c  reduction assay to measure extracellular O²− production by activated hPMNs as described previously. 14,15O²− generation was measured spectrophotometrically as the superoxide dismutase (SOD)-inhibitable reduction of cytochrome c  (fig. 1).

Superoxide anion production was measured over time by the absorbance of cytochrome c  at 550 nm. The reaction was performed in a spectrophotometer (Genesys 5, Spectronic Instruments, Rochester, NY). The reaction mixture was prepared by placing 700 μl buffer (HBSS + bovine serum albumin 0.1%), 200 μl of hPMN suspension (final concentration, 10⁶cells/ml), and 100 μl cytochrome c  (from horse heart, 3.7 mg/ml) with catalase (0.14 mg/ml) in a 1-ml cuvette. The reference sample was prepared the same way, but in addition, 10 μl SOD (10⁻²m) was added to the mixture. Many electron donors in addition to O²− can reduce cytochrome c , but only O²− is destroyed by SOD, by catalyzing its conversion to H²O². By subtracting the absorbance of the SOD reaction (representing cytochrome-c  reduction caused by other radical oxygen metabolites), the selective contribution of O²− can be determined. Catalase was used to degrade H²O²into H²O and O², preventing H²O²from reoxidizing the reduced cytochrome c . Such H²O²-dependent oxidation of reduced cytochrome c  would give inaccurate, falsely low results.

The reaction was activated by either adding N -formylmethionine-leucyl-phenylalanine (fMLP) or phorbol myristate acetate (PMA), and the change in absorbance at 550 nm was monitored over time. The reference sample was measured immediately after, and O²−-dependent cytochrome-c  reduction was determined by subtracting the reference value from the study sample value. To test LA effects on either priming or activation, hPMNs were incubated for 60 min at 37°C in various concentrations of different LAs before hPMN activation with fMLP. When PAF was used as the priming agent, hPMNs were incubated for 5 min before activation with fMLP or PMA, since priming effect by PAF has been shown to be maximal after 5 min. 16Using fMLP for activation, O²− generation at time points 0, 5, 10, 14, 16, and 18 min was measured, because we determined in a previous study maximal activation after 16 min. 3,4PMA-induced superoxide anion production was determined at time points 0 to 50 min every 5 min. O²− production was calculated using as conversion factor 47.4 μmol (1/21.1 mm⁻¹difference of extinction coefficient between oxidized and reduced cytochrome c  at 550 nm) O²− per unit change in absorbance.

We measured extracellular (rather than total) PMN-mediated release of O²− because it is the release of oxygen metabolites into the extracellular milieu that may directly damage cells in the surrounding microenvironment. Extracellular O²− release can be monitored by either end-point or kinetic assays. In spectrophotometric end-point assays, the total amount of O²− that is released into the extracellular environment by a fixed number of PMNs is quantitated after a given incubation time. A kinetic assay determines both the total amount of O²− produced and the rate of O²− release over time. We used a kinetic assay because the kinetics of PMN O²− release are nonlinear and vary with different priming and activating stimuli.

Hank’s balanced salt solution (without phenol red, with Ca²⁺–Mg²⁺) was obtained from Whittaker Bioproducts (Walkersville, MD), and SOD (from bovine liver), fMLP, cytochrome c  (from horse heart), catalase (from bovine liver), PMA, U73122, bisindolylmaleimide, chelerythrine, and pertussis toxin (PTX) were obtained from Sigma Chemicals Co. (St. Louis, MO). Ficoll-Hypaque and bovine serum albumin (protease free bovine albumin fraction–fatty acid free) were obtained from ICN Biomedicals, Inc. (Aurora, OH). Polymorph (Westbury, NY) neutrophil isolation medium was obtained from Cardinal Associates (Santa Fe, NM). PAF (1-alkyl-2-acetoyl-sn -glycero-3-phosphocholine) was obtained from Avanti polar lipids (Alabaster, AL). Lidocaine and QX 314 were gifts from AstraZeneca Pharmaceuticals LP (Westborough, MA).

Data are reported as mean ± SD. Leukocyte metabolic activity is reported either as superoxide anion production or as percentage change from control. Blood from at least eight donors was used for each data point. Groups were compared using either paired Student t  test or one-way repeated-measurement analysis of variance, followed by Dunnett or Student-Newman-Keuls correction (as described in Results), if necessary. P < 0.05 was considered significant. SigmaStat 2.0 (Jandel Scientific Corporation, San Rafael, CA) was used for all statistical analyses.

Because hPMNs were to be incubated in LA for significant duration before the activation assay, we determined, in pilot experiments, the effect of incubation in buffer (37°C, 0–60 min) as well as the effect of movement (by a shaking water bath) on O²− production. Neither affected O²− production as compared with untreated control hPMNs (data not shown). We also determined any interference of PAF with the cytochrome c  assay. PAF did not have any significant effect on absorbance in a neutrophil-free solution as compared with control (data not shown).

After these control experiments, we determined the ability of PAF to activate and/or prime hPMNs. hPMNs were added to the assay solution, and baseline activity was measured to exclude activating effects of the isolation process itself. Activator was then added to the samples, and absorbance was measured for the next 20 min. PAF in concentrations between 10⁻⁴and 10⁻⁸m induced virtually no activation. In contrast, fMLP (10⁻⁶m, a concentration selected as it gave optimal responses in our previous studies 3,4) induced superoxide anion production of approximately 1.9 ± 0.6 nmol/10⁶cells, confirming the functionality of the assay (fig. 2A). When pretreated for 5 min with various PAF concentrations (10⁻⁵m to 10⁻⁸m), responses to fMLP were increased significantly (fig. 2B), indicating that hPMNs could be primed appropriately by PAF, as described previously. 16,17Maximal priming effect (11.1 ± 3.9 nmol/10⁶cells, an almost sixfold increase) was obtained with 10⁻⁶m PAF. Therefore, this concentration was used for the remainder of the study.

We studied the intracellular signaling pathways involved in PAF-induced priming. Because both PAF and fMLP act through G protein–coupled receptors, we determined the effects of PTX on PAF-primed–fMLP-activated O²− production. Preincubation with PTX (0.3 μg/ml) for 90 min caused a modest but significant (P = 0.001, t  test) reduction in superoxide anion production to 73.4 ± 9.6% of control response (13.4 ± 3.1 nmol/10⁶cells;fig. 2C). This suggests that Gⁱand/or G^oproteins are involved but are not the only G-protein subtypes mediating O²− production.

We also investigated the effect of PTX on O²− production of hPMNs primed with PAF (10⁻⁶m) and activated with the PKC agonist PMA, bypassing the G-protein step of the activation pathway. As illustrated in figure 2D, O²− production in response to 1 nm PMA (4.7 ± 1.1 nmol/10⁶cells) increased significantly after incubation with PAF for 5 min (14.4 ± 2.8 nmol/10⁶cells). In contrast to our findings using fMLP as activator, we observed no significant effect of PTX on PAF-primed–PMA-activated O²− production (PTX, 14.1 ± 0.4 nmol/10⁶cells; control, 13.9 ± 0.4 nmol/ 10⁶cells, n = 19;P = 0.105, t  test;fig. 2E). Based on these experiments, we conclude that activation is partially PTX-sensitive, but only PTX-insensitive G proteins are required for the priming process.

Because we determined that PAF priming is mediated through PTX-insensitive G proteins (presumably G^q/11), we studied if priming depends on activation of PLC, the main effector activated by these G proteins.

We primed hPMNs with PAF and measured superoxide anion production after agonist stimulation in the absence and presence of the putative PLC antagonist 1-(6-[([17β]-3-methoxyestra-1,3,5[10]-trien-17-yl)amino]hexyl)-1H-pyrrole-2,5-dione (U-73122). Because the activation pathway may also partly involve PLC, 15we used PMA as the activating stimulus. The priming effect of PAF was abolished completely (5.0 ± 1.9 nmol/10⁶cells) when hPMNs were preincubated for 60 min in U-73122 (10⁻⁶m;fig. 2F), indicating that PLC mediates PAF priming.

Diacylglycerol, generated by the action of PLC on phosphatidylinositolbisphosphate, is the endogenous activator of PKC. Because PAF priming is mediated through a PLC mechanism, we hypothesized that PKC activation plays a role in this pathway. We therefore investigated whether PKC inhibitors block PAF priming. Sixty-minute pretreatment of hPMNs with one of two different PKC inhibitors completely abolished the priming effect by PAF and reduced response sizes to those obtained with fMLP alone (9.5 ± 4.8% of primed response;fig. 3A). Bisindolylmaleimide (10⁻⁶m) reduced superoxide anion production to 12.9 ± 3.6% of control response, and chelerythrine (10⁻⁵m) inhibited O²− production to 5.9 ± 3.1% of control response. No significant difference (P > 0.05, one-way analysis of variance, Dunnett correction) between fMLP-induced responses and those elicited after priming–activation in the presence of bisindolylmaleimide or chelerythrine was obtained. This finding suggests that, in contrast to the activation process, 14priming through PAF is critically dependent on PKC.

If priming is dependent on PKC, one should be able to induce priming by direct PKC activation. At the same time, PMA is able to induce superoxide anion production (although it has been shown that PKC is not required for activation of hPMNs 14). Therefore, it seems likely that PMA treatment of hPMNs would both prime and activate the cells. To determine the maximal priming–activating dose of PMA, we performed a concentration–response study. After adding PMA to hPMNs, superoxide anion production was measured for 40 min (because pilot experiments determined this as sufficient time for O²− production to plateau after PMA stimulation; data not shown). As indicated in figure 3B, maximal superoxide anion production was induced by 100 nm PMA (23.6 ± 4.5 nmol/10⁶cells). Using this PMA concentration to provide maximal stimulation, we next tested our hypothesis that PMA treatment of hPMNs would both prime and activate the cells. We determined first the time course of O²− production after stimulation with PMA (100 nm) and compared it with that obtained after PAF–fMLP treatment. As shown in figure 3C, PMA induced a slower respiratory burst than did PAF–fMLP. Thirty minutes after PMA administration, superoxide anion production reached the maximal level. At this time point, measured O²− production was 174.6 ± 16.6% of the response (13.6 ± 1.6 nmol/10⁶cells) obtained after stimulation with fully priming and activating concentrations of PAF and fMLP. The slow time course suggests that priming may be taking place at the same time as activation.

If PMA indeed acts as a full priming agent, PAF priming of PMA responses might accelerate the onset of priming but should not increase the maximal response. Therefore, we tested the ability of PAF (10⁻⁶m) to prime PMA (100 nm)-induced O²− production. Although PAF accelerated the time course of superoxide production by PMA (fig. 2D), after 30 min no significant difference between O²− production induced by PMA with or without PAF pretreatment was present (fig. 3C). These finding suggests that after 30 min, superoxide anion production by PMA results from a combined priming–activating effect, most likely because of a dual action of PKC on the priming and activation pathways.

After mapping the PAF priming pathway, we studied the effects of LA on priming. We confirmed the findings from our previous study (Durieux ME, unpublished observation, 2000), showing that LAs (lidocaine, tetracaine, and the permanent charged lidocaine analog QX314) do not affect the activation process of hPMNs (data not shown). We then investigated the effect of the most commonly used amide LA, lidocaine (fig. 4A), and the ester LA tetracaine (fig. 4B) on hPMN priming by PAF. hPMNs were incubated for 60 min in various concentrations (10⁻⁴m to 10⁻⁶m) of the LAs, before priming with PAF. Both LAs inhibited superoxide anion production in a modestly concentration-dependent manner over the range tested. Even at an LA concentration (10⁻⁶m) commonly attained in plasma after epidural or intravenous administration, 18–21O²− production was attenuated significantly (to 73.5 ± 6.7% of control response for lidocaine and 68 ± 12.1% of control response for tetracaine;P < 0.05, one-way repeated-measurement analysis of variance, Dunnett correction) as compared with the PAF (10⁻⁶m)-primed–fMLP (10⁻⁶m)-activated control response (11.8 ± 2.5 nmol/10⁶cells for lidocaine and 14.2 ± 7.3 nmol/10⁶cells for tetracaine experiments). At the highest concentration tested (10⁻⁴m), tetracaine (42.8 ± 13% of control response) was found to inhibit O²− production more effectively (P = 0.001, unpaired t  test) than lidocaine (62.8 ± 5.1% of control response).

Because the amide LA ropivacaine has received significant interest as an inflammatory modulator and has undergone clinical studies of its use in inflammatory bowel disease, 22we determined its action on PAF (10⁻⁶m)-primed–fMLP (10⁻⁶m)-activated hPMNs.Both stereoisomers of ropivacaine (10⁻⁴m) inhibited PAF priming with a modest but significant (P < 0.05, one-way repeated-measurement analysis of variance, Student-Newman-Keuls correction) difference between S  (−)-ropivacaine (78.8 ± 3.6% of control response) and the R  (+)-isomer (67 ± 5.1% of control response;fig. 4C).

In addition, we tested the inhibitory potency of bupivacaine, which is structurally similar to ropivacaine but is much more lipophilic. At a concentration of 10⁻⁴m, bupivacaine showed a weak (81.4 ± 4.3% of control response) but nonetheless significant (P < 0.05, paired t  test) inhibition of superoxide anion production compared with PAF (10⁻⁶m)-primed–fMLP (10⁻⁶m)-activated control cells (13.2 ± 4.2 nmol/10⁶cells;fig. 4C). Ropivacaine and bupivacaine were similar in their inhibitory effect (P = 0.06, t  test [n = 18]). Both compounds showed a lower inhibitory potency on priming than did lidocaine or tetracaine, and their effect appears not to depend on lipophilicity.

To obtain additional information about the site of LA action on PAF-induced priming, we tested the effects of the permanently charged and therefore membrane-impermeant lidocaine analog QX314, and the permanently uncharged membrane permeant LA benzocaine. We compared both with the effect of lidocaine on PAF (10⁻⁶m)-primed–fMLP (10⁻⁶M)-activated hPMNs (fig. 4D). Benzocaine (10⁻⁴m) inhibited priming profoundly (to 33.2 ± 4.5% of control response), whereas QX314 (10⁻⁴m) had a modest effect (86.4 ± 6.7% of control response). Lidocaine, as a partially charged compound, was intermediate in effect and inhibited to 59.3 ± 5.9% of control response.

For comparative purposes, we also determined the effects of QX314 and benzocaine on lysophosphatidate (10⁻⁴m) priming in neutrophils (fig. 4D). The findings were very similar to those obtained using PAF. Benzocaine (10⁻⁴m) inhibited primed responses to 39.6 ± 4.8% of control response, whereas QX314 was without effect (101 ± 13.6% of control response). The latter finding is in agreement with our previous observation showing that lysophosphatidate signaling in Xenopus  oocytes is not affected by extracellularly applied QX314. 23Lidocaine inhibited to 69 ± 5.6% of control.

These findings suggest that lysophosphatidate and PAF signaling in hPMNs are affected similarly by LAs. There appears to be, at best, a limited role for extracellular block by charged compounds. The major site of action, therefore, is either an uncharged extracellular domain or an intracellular site, which may be charged or uncharged.

To determine the site of action of lidocaine (10⁻⁴m) in more detail, we studied its effect on hPMNs primed and activated by PMA. We studied the effects 30 min after PMA application, at which time both priming and activation are complete (fig. 3B). As shown in figure 5A, lidocaine was without effect when cells were stimulated with 100 nm PMA (lidocaine, 21.7 ± 0.5 nmol/10⁶cells; control, 22 ± 0.8 nmol/10⁶cells; n = 17;P = 0.2, t  test) or 10 nm PMA (lidocaine, 13.1 ± 0.4 nmol/10⁶cells; control, 13.3 ± 0.4 nmol/10⁶cells; n = 20;P = 0.122, t  test). Thus, lidocaine did not affect PMA-induced O²− production, suggesting that neither in activation nor priming pathway is its target site located downstream of PKC.

Finally, we determined if inhibition of Gⁱ–G^omay play a role in the effects of lidocaine. We preincubated hPMNs in PTX (0.3 μg/ml) and lidocaine (10⁻⁴m). As demonstrated in figure 5B, superoxide anion production was reduced to 31.4 ± 7.9% of control response. Lidocaine still exerted an approximately 40% inhibition of PAF (10⁻⁶M)-primed–fMLP (10⁻⁶m)-activated hPMNs in addition to the 31% inhibition by PTX. This is similar to its effect in the absence of PTX (fig. 4A). Taking this finding together with the previously described lack of lidocaine inhibition on fMLP-induced activation, the main target site for lidocaine is likely to be different from G^i/o.

In the current study, we have shown that PAF primes neutrophils through a pathway dependent on PTX-insensitive G proteins, PLC, and PKC. PKC activation is both necessary and sufficient for this process. In addition, we show that clinically relevant concentrations of LAs selectively inhibit priming but not fMLP-induced activation. Ester LA exerted the most profound inhibitory effect, whereas inhibitory potency of amide LA increased with increased uncharged fraction. The main target site for LAs in the PAF priming pathway is located upstream of PKC.

The concentrations at which these effects take place are much less than those required to block sodium channels. Lidocaine showed significant inhibitory effects on priming, even at concentrations commonly obtained in plasma of patients after epidural or intravenous administration (approximately 0.5–5 μg/ml, corresponding to 2–20 μm) 18; for example, intravenous administration of lidocaine at 2–4 mg/min leads to plasma concentrations of 1–3 μg/ml (4–12 μm) after 150 min. 19A 2-mg/kg intravenous bolus dose of lidocaine results in peak plasma levels of 1.5–1.9 μg/ml (6–8 μm) after 15 min. 20Similar plasma concentrations are obtained after epidural administration. 21The effects of LAs on priming of hPMNs might be one explanation for the discrepancy between concentrations required to achieve antiinflammatory effects of LA in vitro  and in vivo . Virtually all in vitro  studies of LA effects on hPMNs investigated activation. To our knowledge, the effects of LAs on priming have not been reported previously, although this state of hPMNs should be more representative for in vivo  inflammatory processes. Primed hPMNs have been identified in the peripheral blood of patients after blunt trauma, 24acute respiratory distress syndrome, 25and bacterial or fungal infections. 26hPMN priming has been shown to be critical for the induction of endothelial injury 27and lung damage 28 in vivo . Because LAs, in reasonable concentrations, did not block unprimed neutrophil metabolic activity, this might explain why antiinflammatory LA concentrations required to block activation in vitro  are generally much greater than those required to block inflammatory responses in vivo . 1,2Another factor may be that duration of LA incubation (60 min) is longer in our study than in most other studies. We believe that this mimics the clinical setting more closely.

We used different LAs to determine which chemical properties might enhance or reduce their inhibitory potency. Because tetracaine and benzocaine (ester LA) showed inhibition comparable to or even more profound than that obtained with amide LA, the inhibitory effect appears independent of the linkage type within the LA molecule, consistent with our findings on lysophosphatidate-primed hPMNs (Durieux ME, unpublished observation, 2000). For the amide LA lidocaine (ropivacaine, bupivacaine, and QX314), we observed an increase in inhibitory potency with increased uncharged fraction. Lidocaine, a representative mostly uncharged amide LA at physiologic pH, exerted the most profound inhibition within the group of amide compounds, whereas bupivacaine (highly lipophilic and highly charged at pH 7.4) and QX314 (permanently charged) showed the least inhibitory effect. Obviously, these findings may reflect a need for the compound to traverse the cell membrane to reach its site of action. Ropivacaine, which is structurally similar to bupivacaine but similar in lipophilicity to lidocaine, showed no significant difference in inhibitory potency as compared with bupivacaine, suggesting that the inhibitory effect is independent of lipophilicity of the compound.

The site of local anesthetic action is of obvious interest. Interaction with sodium channels is unlikely, as these are not present in hPMNS, 29and concentrations used are less than those required to block sodium channels. Lidocaine failed to inhibit PMA-induced superoxide anion production, indicating that the major site of action of lidocaine has to be located upstream of PKC in the PAF priming pathway. The effects of LAs on the activation process were excluded in this and also in our previous study (Durieux ME, unpublished observation, 2000). The site of action is therefore likely to be located between receptor and PKC. In Xenopus  oocytes, we previously showed that LAs do not interfere with the PLC–inositol trisphosphate–calcium pathway. 23If these findings can be extrapolated to hPMNs, the PAF receptor or G protein(s) itself seem to be the most likely target. QX314 was virtually without effect when applied extracellularly, excluding a charged extracellular site as the main target. However, an uncharged extracellular site cannot be ruled out. In oocytes, we have shown intracellular QX314 to inhibit G^qproteins selectively. 30Because we determined that PAF priming is mediated through a PTX-insensitive G protein, we hypothesize that G^qis a likely LA target in inhibiting PAF priming. This is in agreement with the finding that lidocaine still exerted its inhibitory action in the presence of PTX and with previous findings from our group showing that neither Gⁱ(Durieux ME, unpublished observation, 2000) nor G^o30signaling is inhibited by relevant LA concentrations. Although G^s-mediated signaling is PTX-insensitive, it is unlikely to be involved because increases in cyclic adenosine monophosphate levels, resulting from G^sactivation, inhibit neutrophil functions. 31–33 

Protein kinase C activation with PMA is able to both prime and activate hPMNs. PMA induces respiratory burst in a concentration-dependent manner. We determined that PMA-induced superoxide anion production could be primed effectively with PAF after 15 min. However, maximal PMA-induced O²− production obtained after 30 min is most likely the result of a combined priming and activation effect, as PAF is no longer able to increase O²− production further. Lack of priming effect on PMA-stimulated superoxide anion production by PAF after 30 min cannot be attributed to loss of PAF action, because its priming effect on fMLP-induced O²− production did not change over a 50-min time course.

Using different PKC inhibitors (including the broad-range PKC inhibitor bisindolylmaleimide), we demonstrated that PAF priming is inhibited completely by PKC blockade, indicating that the priming process is critically dependent on PKC. This is in contrast to activation by fMLP: Pongracz et al. 14showed that several PKC inhibitors were not able to affect fMLP-induced superoxide anion production.

Pertussis toxin partially inhibited superoxide anion production in PAF-primed, fMLP-activated hPMNs but not in PAF-primed, PMA-activated cells. hPMNs were incubated in PTX for 90 min before their activation, as reported by Christiansen. 15We used a PTX concentration of 0.3 μg/ml when studying fMLP activation, as this concentration has been shown to inhibit fMLP-induced superoxide anion production by approximately 50%. 15Higher concentrations would have abolished fMLP activation completely and were therefore used only when studying PAF-primed–PMA-activated cells. Despite the greater concentration, PTX was without effect on PAF-primed, PMA-activated hPMNs. The most likely explanation is that PTX partially inhibited fMLP-induced activation but was without effect on priming. In fact, several investigators reported that fMLP-induced signaling in hPMNs is mediated by PTX-sensitive G proteins. 34–36Our hypothesis of the signaling pathways involved in superoxide anion production of PAF-primed and fMLP-activated hPMNs is illustrated in figure 6.

In conclusion, our data provide new insights into the mechanism of hPMN priming and, in addition, suggest a mechanism by which LAs may exert some of their antiinflammatory actions.

The authors thank Prof. Dr. med. Eike Martin, M.D., Ph.D. (Ruprecht-Karls-Universität, Heidelberg, Germany), for his support, and AstraZeneca Pharmaceuticals LP (Westborough, MA) for supplying QX314 and stereoisomers of ropivacaine.