Inhibitory Effects of Intravenous Anesthetics on Mast Cell Function

Takahiro Fujimoto, MD, PhD, Tomoki Nishiyama, MD, PhD, and Kazuo Hanaoka, MD, PhD
Department of Anesthesiology, Graduate School of Medicine, The University of Tokyo, Tokyo, Japan.

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Abstract

Mast cells play a protective role in the inflammation and auto-tissue injury. The impairment of mast cell function may influence defense against infection. We investigated the effect of four intravenous anesthetics (thiopental, midazolam, ketamine and propofol) on chemotaxis and exocytosis of mast cells. Canine mast cells chemotaxis was measured by the Boydenﾕs blindwell chamber technique using 100_g/ml of substance P as a stimulator. We measured mast cell exocytosis by measuring released histamine from mast cells using substance P or ganmma-monomeric IgG-mediated crosslinking as a stimulator. Thiopental, midazolam and propofol exerted a dose dependent inhibitory effect on mast cell chemotaxis. Ketamine, midazolam and propofol had a dose dependent inhibitory effect on mast cell exocytosis. In conclusion, midazolam and propofol inhibited both chemotaxis and exocytosis of mast cell, respectively, while thiopental did only chemotaxis and ketamine did only exocytosis.
Key word: mast cell; substance P; chemotaxis; exocytosis; midazolam; propofol; thiopental; ketamine
Implications
Mast cell plays an important role in antibacterial host defense mechanism. Thiopental, midazolam and propofol exerted a dose dependent inhibitory effect on mast cell chemotaxis. Ketamine, midazolam and propofol had a dose dependent inhibitory effect on mast cell exocytosis. The impairment of mast cell function by intravenous anesthetics may influence defense against infection.

Introduction

General anesthesia has been considered to influence immune function. The inhibitory effects of thiopental, ketamine and midazolam on neutrophil functions have been well documented (1), while there are no reports of the effects of anesthetics on mast cell function. Mast cells are distributed in various tissues, and affect local microcirculation in inflammation (2). Mast cells are now recognized as an important source of mediators, multifunctional cytokines. Recent evidence suggests that the degranulation of mast cells induce granulocyte (neutrophil and eosinophil) infiltration into inflammatory skin (3), which may play a important protective role in inflammation (2,3). Mast cell dysfunction may loose the defense to bacterial infection.
In the present study, first, we evaluated the potency of chemoattractants for canine mast cell. We have examined laminin, fibronectine, compound 40/80, calcium ionophore (A23187) and Substance P, the biologic active substances which induce canine mast cell degranulation (4). Second, we investigated the effect of four intravenous anesthetics (thiopental, midazolam, ketamine and propofol) on chemotaxis and exocytosis of mast cells.

Methods

Drugs
Substance P, calcium ionophore A23187, laminin, fibronectin were obtained from (Sigma Chemical Co.,St.Louis,Mo.,USA). Compound 48/80 were obtained from (Peptide Institute Inc.,Osaka,Japan). Canine IgG (10_g /ml, ICN Pharmaceuticals Inc., Aurora, OH, USA). Anti-canine IgG were obtained from (10 _g/ml, Bethyl, Montgomery, TX, USA)Thiopental were obtained from (Ravonalｨ; Tanabe, Osaka, Japan). Ketamine were obtained from (Ketalarｨ; Sankyo, Tokyo, Japan). Midazolam were obtained from (Dormicumｨ; Yamanouchi, Tokyo, Japan). Propofol were obtained from (Diprivanｨ; Astrazeneka, Osaka, Japan)

Cells and buffers
Neoplastic mast cells were obtained from a dog with cutaneous mastocytoma (CMMC). This cell population was characterized morphologically and functionally, and has been used as a useful mast cell model. IgE- and substance P- mediated histamine release from CMMC (4), the expression of Fc_（crystallizable fragment _）receptors on the cell, monomeric IgG-binding to the cell and IgG- or IgE-mediated signal transduction in CMMC (5) have already been reported. Following a published protocol (4, 5), the CMMC were passaged in RPMI 1640 medium (Irvine Scientific, Santa Ana, CA) with 10% heat-inactivated fetal calf serum. The Pipes-ACM buffer used for the experiments consisted of 140mM NaCl, 5mM KCl, 0.6mM MgCl2, 1mM CaCl2, 5.5mM glucose, 0.1% Bovine Serum Albumin and 10mM Pipes A (pH 7.4). All concentrations of substance P were nontoxic for CMMC as assessed by trypan blue.
Canine mast cells chemotaxis analysis
Canine mast cells chemotaxis was measured by a modified Boydenﾕs blindwell chamber technique (6) using a 48-well chamber (7-11), and an 8-_m-pore-size membrane (polyvinylprolidine free) (Neuroprobe, Gaithersburg, MD. USA). various concentrations of chemotactic agents (laminin, fibronectine, compound 40/80, calcium ionophore (A23187) and Substance P(0,30,100,300_M) was placed in the bottom compartment of the assay chamber and covered with a framed filter. The top compartment, with a silicone gasket, was carefully set in place, and the wing nuts were tightened. The filter sheet was wetted with Pipes ACM. Then 50_l of cell suspension which contained 1.5_106 canine mast cells was placed in each upper well of the top compartment. The chamber was incubated at 37℃, in a humidified atmosphere containing 5% CO2 for 2h. The filter was removed and cells remaining on the top wells were removed by scraping. The membrane-bound cells were carefully washed with buffered saline and stained with Diff-Quick stain (American scientific products, McGaw Park, IL, USA). The number of cells that had migrated were counted in 10 high power fields (HPF) using a light microscope (BX51, Olympus, Osaka, Japan) at_400 magnification. Cell migration was calculated as the average number of 10 HPF per well. The time required for canine mast cells chemotaxis to substance P was measured by varying the incubation time of the chemotaxis chambers at 30min, 1,2,3,4 6h.Checkerboard Analysis for Verification of Chemotaxis
To confirm that the cell migration was chemotactic, ﾒcheckerboardﾓ analysis was performed (12). The purpose of this analysis was to discriminate chemotaxis from chemokinesis. In the top wells of the chemotactic chamber, various concentrations of substance P (0, 30, 100, and 300_M) were applied together with the target cells. In the bottom wells, the same concentrations of substance P were placed such that all possible combinations above and below the filter were tested. Cell migration was measured in the presence of various concentration of substance P, both in the presence and absence of gradients of chemoattractant. Each combination was tested in triplicate. The location of the various combinations in the 48-well manifold was randomized.
Canine mast cell exocytosis analysis
We used 100_g/ml of substance P or rmonomeric IgG-mediated crosslinking as a stimulator of canine mast cells exocytosis. Canine mast cells (5 x 105 cells/ml) were sensitized with canine IgG for 30 min at 37℃. After washing twice with Pipes-A buffer, cells were resuspended in Pipes-ACM buffer (3mM). The cells were stimulated with anti-canine IgG at 37℃ and the reaction was terminated 45 min later by centrifugation and the supernatant collected. Histamine concentrations were measured using o-phthalaldehyde fluorometric technique modified for autoanalysis as previously described (9). The percent histamine release (%HR) was calculated by a following formula as previously described (4). [(Histamine release by each agent ﾐ spontaneous histamine release) / (Total histamine content ﾐ spontaneous histamine release)] x 100 (%). All measurements were performed in duplicate
Effects of anesthetics
We evaluated the intravenous anesthetics inhibitory effect on canine mast cells chemotaxis using 100_g/ml of substance P as a stimulator. Canine mast cells were preincubated with various concentrations of four intravenous anesthetics in medium for 120 minutes at 37｡C. The cells were then washed twice and resuspended in complete medium before use in chemotaxis and exocytosis assay. The intravenous anesthetics were diluted in Pipes ACM, and placed in the measuring cuvette to a total volume of 1 or 2 ml, giving the following final concentrations: thiopental and Ketamine 3, 30, and 300 _g/ml, midazolam 0.15, 1.5, and 15 _g/ml, and propofol 0.5, 5, and 50 _g/ml. These concentrations correspond to 0.1, 1 and 10 times of clinically relevant plasma concentrations which were reported by NIshina(18) and Mikawa(19).

Statistics

The results are expressed as meanｱSD. Data were analyzed with repeated measures analysis of variance. A value of P< 0.05 was considered statistically significant.

Results

Canine mast cells Chemotaxis
We first examined the ability of laminin, fibronectin, compound 40/80, substance P, calcium ionophore A23187, canine mast cell in a Boyden chamber assay. Significant mast cell migration could be seen only when substance P was used as a chemotactic agents. (Fig.1) The number of migrated cells per HPF with 100_M of substance P (19.7ｱ 1.5) (Fig. 2A) was significantly larger than that without substance P (1.0 ｱ 0.6) (Fig. 2B). Figure 3A shows a concentration response curve of mast cell migration. Figure 3B shows that the number of canine mast cells increased by increasing incubation time and reached a plateau at 2 h.
Effects of anesthetics
Thiopental, midazolam and propofol had dose dependent inhibitory effects on mast cell chemotaxis (Fig.4). At clinically relevant concentrations, ketamine (30_g/ml=109_M) did not inhibit chemotaxis. Ketamine, midazolam and propofol exerted dose dependent inhibitory effects on mast cell exocytosis (Fig.5). At 0.1, 1 and 10 times clinical relevant plasma concentrations, thiopental (3, 30, 300_g/ml=11.4, 114, 1140_M) did not inhibit exocytosis.

Discussion

In the present study, first, we evaluated the potency of chemoattractants for canine mast cell. We have examined laminin, fibronectine, compound 40/80, calcium ionophore (A23187) and Substance P, the biologic active substances which induce canine mast cell exocytosis (4). Significant mast cell migration could be seen only when substance P was used as a chemotactic agents. Figure 2 and 3A show that cell migration was chemotactic, since migrated cells were increased in the presence of the gradient of substance P concentration.
Mature mast cells are resident cells found in most of the tissues (20). The factors that control migration of mast cells to sites of inflammation and tissue repair remain unknown. In murine mast cells, interleukin-3 (7), stem cell factor (SCF) (8), and transforming growth factor _ (9), were found to promote mast cell migration. In addition, SCF (10) and anaphylatoxins (C3a and C5a) (11) have been reported to induce chemotaxis in human mast cells.
The interaction of allergic Ag and their specific cytotrophic antibodies on the membrane of mast cells induces degranulation of mast cells and release of chemical mediators (21). Some biologic active peptides such as substance P and nerve growth factor have been shown to evoke histamine release from mast cells through their specific receptors (4). Substance P is released from peripheral nerve endings of sensory neurons by various stimuli, and the released substance P induces cutaneous vazodilation, plasma extravasation (3).The observation that mast cells are close to endings of sensory nerves (15) suggests biologic activity as mast cell activator of substance P released from sensory neurons after inflammatory stimulation. (15)
Second, we investigated the effect of four intravenous anesthetics (thiopental, midazolam, ketamine and propofol) on chemotaxis and exocytosis of mast cells. The impairment could affect a patientﾕs ability to fight infection and wound healing. We used substance P as a canine mast cells chemotaxic factor to recruit mast cells. Although there are no reports of mast cell chemotaxis, several studies report intravenous anesthetics inhibited chemotaxis of neutrophils. Skoutelis et al. (17) and Nishina et al. (18) reported inhibitory effect of thiopental on neutrophil chemotaxis. Ketamine at clinically relevant concentration had no depression on neutrophil chemotaxis (18). Nishina et al. (18) reported inhibitory effect of midazolam on neutrophil chemotaxis at clinically relevant concentrations. Skoutelis et al. (17) and Mikawa et al.(19) reported inhibitory effect of propofol on neutrophil chemotaxis. These results on neutrophil chemotaxis were identical to the effects on mast cell chemotaxis in the present study.
We used substance P (which induces Gi-protein activation) and monomeric IgG-mediated crosslinking (which induces protein tyrosine phosphorylation) stimulator of canine mast cells exocytosis. We found that thiopental did not decrease exocytosis on mast cells. There are few reports of direct effects of intravenous anesthetics on human mast cell exocytosis. Stellato et al. (22) reported thiopental induced histamine release from human lung mast cells, but did not induce de novo synthesized mediators (peptide leukotriene C4 (LTC4) or prostaglandin D2 (PGD2)). Stellato used human cutaneous mast cells, while thiopental did not induced histamine. In our study, ketamine, midazolam and propofol exerted a concentration dependent inhibitory effect on canine mast cells exocytosis (Fig.5).
We have shown that at clinically relevant concentrations, intravenous anesthetics suppressed mast cell functions. This suppression and it could decrease self defense to infection, may be more serious for critically ill patient. Thiopental increased pneumonia in ventilated head-trauma patients (23). This report supports our result in vitro.
In conclusion, we have shown that thiopental, midazolam and propofol but not thiopental exerted a dose dependent inhibitory effect on mast cell chemotaxis. Ketamine, midazolam and propofol exerted a dose dependent inhibitory effect on mast cell exocytosis.

References

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Figure Legends

Figure 1
Migration of canine mast cells to substance P (_), fibronectine(_), laminin(_).Bar represents SD for three individual experiments. canine mast cell also did not migrate in response to compound 40/80 and calcium ionophore (A23187) similar to fibronectine and laminin(data not shown).
Figure2　
Canine mast cells migrated through the chemotaxic membrane.
(A) Migrated canine mast cells with substance P (100_M) stained by Diff Quick. (B) Migrated canine mast cells without substance P stained by Diff Quick. Arrow shows migrated cells. Arrowhead shows 8-_m pore of the membrane. The bar in the figure indicates 10-_m scale.
Figure 3
A Dose-dependent chemotaxis of canine mast cells to substance P.
canine mast cells are sensitized with substance P and suspended in Pipes ACM buffer(◆) in Pipes EGTA buffer (absence of extracellular Ca2+)(_).
Vertical axis: canine mast cells chemotaxis expressed as a number of cells migrated per HPF. Horizontal axis: concentration of Substance P. Time point was 2h. Shown are meanｱSD, each assayed for chemotactic activity in tripricate.
B Canine mast cells chemotaxis to substance P (100_M). Time dependence of cell chemotaxis
Vertical axis: canine mast cells chemotaxis expressed as number of cells migrated per HPF. Horizontal axis: incubation time of chemotaxis chamber. Shown are mean±SD, each assayed for chemotactic activity in tripricate.

Figure 4
Effects of thiopental (A), midazolam (B), ketamine (C), and propofol (D) on mast cell chemotaxis. Data (mean ｱSD) are expressed as a percentage of the control (in the absence of intravenous anesthetics). # = clinically relevant concentration of each anesthetic. ＊= P<0.05 versus control.
Figure 5
Effects of thiopental (A), midazolam (B), ketamine (C), and propofol (D) on mast cell exocytosis. Data (mean ｱSD) are expressed as a percentage of the control. # = clinically relevant concentration of each anesthetic. ＊= P<0.05 versus control. Dotted and striped columns indicate substance P and monomeric IgG-mediated crosslinking stimulation, respectively.