General anesthesia may influence immune function. The inhibitory effects of thiopental, ketamine, and midazolam on neutrophil functions have been well documented (1), whereas 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 a new class of inflammatory mediators, known as multifunctional cytokines. Evidence suggests that the degranulation of mast cells induces granulocyte (neutrophil and eosinophil) infiltration into inflammatory skin (3), which may play a important protective role in inflammation (2,3). Mast cell dysfunction may reduce the defense against bacterial infection.
In the present study, we first evaluated the potency of chemoattractants for canine mast cells. We have examined laminin, fibronectin, compound 40/80, calcium ionophore (A23187), and substance P, the biologically active substance that induces canine mast cell degranulation (4). Second, we investigated the effect of four IV anesthetics (thiopental, midazolam, ketamine, and propofol) on the chemotaxis and exocytosis of mast cells.
Substance P, calcium ionophore (A23187), laminin, and fibronectin were obtained from Sigma Chemical (St.Louis, MO). Compound 48/80 was obtained from Peptide Institute Inc. (Osaka, Japan). Canine IgG (10 μg/mL) was obtained from ICN Pharmaceuticals Inc. (Aurora, OH). Anti-canine IgG (10 μg/mL) was obtained from Bethyl (Montgomery, TX). Thiopental (Ravonal®) was obtained from Tanabe (Osaka, Japan). Ketamine (Ketalar®) was obtained from Sankyo (Tokyo, Japan). Midazolam (Dormicum®) was obtained from Yamanouchi (Tokyo, Japan). Propofol (Diprivan®) was obtained from Astrazeneka (Osaka, Japan).
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 Fcγ (crystallizable fragment γ) receptors on the cell, monomeric IgG-binding to the cell, and IgG- or IgE-mediated signal transduction in CMMC (5) have been reported. Following a published protocol (4,5) the CMMC was processed 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 140 mM NaCl, 5 mM KCl, 0.6 mM MgCl2, 1 mM CaCl2, 5.5 mM glucose, 0.1% bovine serum albumin, and 10 mM Pipes A (pH 7.4). All concentrations of substance P were nontoxic for CMMC as assessed by trypan blue.
Canine mast cell chemotaxis was measured by a modified Boyden's blindwell chamber technique (6) using a 48-well chamber (7–11) and an 8-μm pore-size membrane (polyvinylprolidine free) (Neuroprobe, Gaithersburg, MD). Various concentrations of chemotactic agents laminin, fibronectin, compound 40/80, calcium ionophore [A23187], and sbstance P (0, 30, 100, 300 μM) were 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 μL of cell suspension that contained 1.5 × 106 canine mast cells was placed in each upper well of the top compartment. The chamber was incubated at 37°C, in a humidified atmosphere containing 5% CO2 for 2 h. 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). 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 maginification. 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 30 min, and 1, 2, 3, 4, and 6 h).
To confirm that the cell migration was chemotactic, “checkerboard” 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 μM) 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 concentrations 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.
We used 100 μg/mL of substance P or monomeric IgG-mediated crosslinking as a stimulator of canine mast cells exocytosis. Canine mast cells (5 × 105 cells/mL) were sensitized with canine IgG for 30 min at 37°C. After washing twice with Pipes-A buffer, cells were resuspended in Pipes-ACM buffer (3 mM). The cells were stimulated with anti-canine IgG at 37°C, 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 was calculated by the following formula, as previously described (4):
All measurements were performed in duplicate.
We evaluated the IV anesthetics' inhibitory effect on canine cell chemotaxis using 100 μg/mL of substance P as a stimulator. Canine mast cells were preincubated with various concentrations of four IV anesthetics in medium for 120 min at 37°C. The cells were then washed twice and resuspended in complete medium before use in chemotaxis and exocytosis assay. The IV 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 μg/mL; midazolam 0.15, 1.5, and 15 μg/mL; and propofol 0.5, 5, and 50 μg/mL. These concentrations correspond to 0.1, 1, and 10 times of clinically relevant plasma concentrations that were reported by Nishina et al. (13) and Mikawa et al. (14).
The results are expressed as mean ± sd. Data were analyzed with repeated measures analysis of variance. A value of P < 0.05 was considered statistically significant.
Significant mast cell migration was only observed when substance P was used as a chemotactic agent (Fig. 1). The number of migrated cells per HPF with 100 μM of substance P (19.7 ± 1.5) (Fig. 2A) was significantly larger than that without substance P (1.0 ± 0.6) (Fig. 2B). Figure 3A shows a concentration response curve of mast cell migration. Fig. 2B shows that the number of canine mast cells increased by increasing incubation time and reached a plateau at 2 h.
Thiopental, midazolam, and propofol had dose-dependent inhibitory effects on mast cell chemotaxis (Fig. 4). At clinically relevant concentrations, ketamine (30 μg/mL = 109 μM) 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 μg/mL = 11.4, 114, 1140 μM) did not inhibit exocytosis.
In the present study, we first evaluated the potency of chemoattractants for canine mast cells. We have examined laminin, fibronectin, compound 40/80, calcium ionophore (A23187), and substance P, the biologically 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 agent. Figures 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 tissues (15–18). The factors that control migration of mast cells to sites of inflammation and tissue repair are unknown. In murine mast cells, interleukin-3 (7), stem cell factor (SCF) (8), and transforming growth factor β (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 antigens and their specific cytotrophic antibodies on the membrane of mast cells induces degranulation of mast cells and release of chemical mediators (19). Some biologically 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 vasodilation, and plasma extravasation (3). Substance P is released from sensory neurons (20) with inflammatory stimulation. The observation that mast cells are close to endings of sensory nerves suggests that substance P promotes biologic activity of mast cells (20).
Second, we investigated the effect of four IV anesthetics (thiopental, midazolam, ketamine, and propofol) on chemotaxis and exocytosis of mast cells. The impairment could affect a patient's ability to fight infection and wound-healing. We used substance P as a canine mast cell chemotaxic factor to recruit mast cells. Although there are no reports of mast cell chemotaxis, several studies have reported that IV anesthetics inhibited chemotaxis of neutrophils. Skoutelis et al. (21) and Nishina et al. (13) reported the inhibitory effect of thiopental on neutrophil chemotaxis. Ketamine, at clinically relevant concentrations, had no depression on neutrophil chemotaxis (13). Nishina et al. (13) reported the inhibitory effect of midazolam on neutrophil chemotaxis at clinically relevant concentrations. Skoutelis et al. (21) and Mikawa et al. (19) reported the 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) as stimulators of canine mast cell exocytosis. We found that thiopental did not decrease exocytosis on mast cells. There are few reports of direct effects of IV anesthetics on human mast cell exocytosis. Stellato et al. (22) reported that thiopental induced histamine release from human lung mast cells, but did not inducede novosynthesized mediators (peptide leukotriene C4 [LTC4] or prostaglandin D2 [PGD2]). Stellato et al. (22) used human cutaneous mast cells, while thiopental did not induce histamine. In our study, Figure 5 shows that ketamine, midazolam, and propofol exerted a concentration-dependent inhibitory effect on canine mast cell exocytosis.
We have shown that, at clinically relevant concentrations, IV anesthetics suppressed mast cell function. This suppression could decrease the body's defense against infection, and may be more serious for critically ill patients. Thiopental increased pneumonia in ventilated head-trauma patients (23).This report supports our resultin vitro.
In conclusion, thiopental, midazolam, and propofol, but not ketamine, exerted a dose-dependent inhibition of mast cell chemotaxis
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