If mast cells are stimulated they release multiple mediators that delineate markers for immunologic and nonimmunologic reactions; histamine and tryptase are the two best known. Although histamine can be assayed in plasma, it is a nonspecific marker with a very short half-life. Tryptase has a longer half-life, but its release has not been proven to be specific for anaphylaxis. The authors investigated the mechanisms of nonimmunologic histamine release from human cutaneous mast cells to understand the mechanisms of mediator release and to determine whether tryptase was specific for allergic mediated activation.
Dispersed mast cell suspensions isolated from neonatal foreskins underwent challenge with vancomycin, calcium ionophore A23187, morphine, and atracurium, and histamine tryptase release was measured. The effects of calcium and magnesium, along with phospholipase C and phospholipase A2 inhibitors, also were investigated.
Tryptase and histamine both were released by the known nonimmunologic stimuli (pharmacologic agents used in the current study; r2 = 0.6). Furthermore, vancomycin- and atracurium-induced histamine release was calcium dependent. Phospholipase C and phospholipase A2 inhibitors decreased vancomycin-induced histamine release, but not calcium ionophore A23187-induced release.
Tryptase is not a specific marker of mast cell activation (ie., anaphylaxis), and signaling mechanisms for mast cell activation involve activation of phospholipase C and phospholipase A2 pathways that are also involved in other cellular activation mechanisms.
ANAPHYLACTIC reactions represent complex acute inflammatory processes that are associated with significant morbidity and mortality rates. A key inflammatory cell in this clinical event is the mast cell. Mast cells can release histamine and tryptase through multiple mechanisms, including immunologic (by antigens) or nonimmunologic (by drugs or neurokinins) stimulation. Immunologic activation initiates the release of histamine and other mediators by a mechanism that takes 5 min to reach completion and needs influx of extracellular calcium. 1,2Although nonimmunologic mast cell activation is not well-understood, vancomycin, 3the calcium ionophore A23187, 4morphine, 5and atracurium 6,7consistently cause degranulation. A common feature of immunologic and nonimmunologic stimulation is the release of intracellular histamine. However, histamine is cleared within minutes from the circulation; in contrast, tryptase stays elevated for 1–2 h or more and appears to be released from mast cells with histamine. Although the physiologic role of tryptase as a mediator is not clear, measurement of tryptase concentrations potentially can provide a better diagnostic tool to evaluate suspected hypersensitivity reactions if sampling is delayed or as a more specific marker for mast cell activation. 8Schwartz et al. 9described that tryptase can be used in vivo as a diagnostic correlate of mast cell activation in systemic anaphylaxis and mastocytosis. However, there is some controversy about whether tryptase is released with histamine, especially if nonimmunologic degranulation occurs. Because tryptase is considered to be a better marker for immunologic activation, we investigated whether human cutaneous mast cells release tryptase with histamine if stimulated nonimmunologically with vancomycin, the calcium ionophore A23187, morphine, or atracurium.
Little information is available about the mechanisms of nonimmunologic mast cell degranulation. An understanding of the nonimmunologic mechanisms of mast cell activation may provide therapeutic approaches to treating or preventing angioedema. Therefore, we investigated whether the mast cell degranulation was calcium dependent. In addition to histamine and tryptase, eicosanoids, such as prostaglandins and leukotrienes that arise from the metabolism of arachidonic acid, are also released after immunologic activation of cutaneous mast cells to produce inflammatory responses. 1,5,10Arachidonic acid production is thought to be associated with an activation of cytosolic phospholipase A2(PLA2) in mast cells. 11Because the calcium ionophore A23187 can activate the Ca2+-dependent cytosolic PLA2, with a resultant increase in arachidonic acid, we evaluated the involvement of cytosolic PLA2. We also evaluated the possibility that arachidonic acid arises via a phospholipase C (PLC) mechanism.
Materials and Methods
Isolation of Neonatal Mast Cells
Human foreskins were obtained after circumcision. The skin was immediately placed in Ca2+–Mg2+-free Hanks’ balanced salt solution (CMF-HBSS; Sigma Chemical, St. Louis, MO) at 4°C and used within 24 h of circumcision. 4,6,12–16After removal of the subcutaneous fat the skin was weighed, chopped into 1- or 2-mm fragments, and washed twice with CMF-HBSS (10 ml/g) at room temperature. The tissue fragments were then incubated in CMF-HBSS with collagenase (20 mg/g), hyaluronidase (4 mg/g), and deoxyribonuclease (1,000 U/foreskin; Worthington Biochemical, Freehold, NJ) in a total volume of 5 ml/g wet weight of tissue for 3 h at 37°C, with constant stirring. After incubation the digested tissue was filtered through a 150-μm pore-sized Nytex cloth (Tetko, Elmsford, NJ) and washed three times with 3.5% bovine serum albumin made in CMF-HBSS (Sigma Chemical).
Dispersed cells were resuspended in RPMI-1640 (Roswel Park Memorial Institute) culture medium (10 ml/g skin) with 25 mM HEPES (Sigma Chemical), 2 mM glutamine, 5% fetal calf serum (GIBCO, Grand Island, NY), 1,000 U/ml penicillin G, and 1% gentamicin (vol/vol; Sigma Chemical). The suspension was inoculated into a Petri dish and incubated overnight at 37°C in humidified 95% air and 5% CO2.
After overnight culture, the cells were harvested and washed three times with PAG buffer (5 ml/g), [Pipes, 25 mM piperazine-N,N′-bis(-ethenesulfonic acid) (Sigma Chemical), 110 mM NaCl, 6 mM potassium chloride (KCL), and 0.1% dextrose, pH 7.4 at room temperature. They then were counted in a hemocytometer after staining with toluidine blue. Viability was assessed by trypan blue exclusion. If the assessed cell viability was less than 80%, the cells were discarded. A Wright-stained Cytospin preparation (Shandon Inc., Pittsburgh, PA) was used to determine the percentage of mast cells in each sample.
Histamine and Tryptase Release after Drug Challenge
One hundred-microliter aliquots of the cell suspension, containing between 2 × 104and 5 × 104mast cells/experimental tube were incubated in duplicate for 30 min at 37°C with the following drugs: 4.76 × 10−5M calcium ionophore A23187 (Sigma Chemical); 3 × 10−3M vancomycin (Vancocin HCl; Lilly, Indianapolis, IN); 10−3M atracurium (Tracurium; Burroughs Wellcome, Triangle Park, NC); and 2.63 × 10−3M morphine (Mallinckrodt, St. Louis, MO). The concentrations chosen were based on in vitro data 3and data applied to clinically relevant doses. 17To determine the effects of calcium on histamine release, cells were incubated either in buffer from which Ca2+and Mg2+were omitted or in buffer containing 2.8 mM Ca2+and 1.0 mM Mg2+.
The release reaction was stopped by addition of 900 μl ice-cold CMF-HBSS and centrifugation at 1,000 g for 5 min at 4°C. Collected supernatants were split into two aliquots and frozen at −70°C until analysis time. The total-release tubes had 900 μl of sterile ice water added instead. Total histamine–tryptase content of the skin mast cells was assessed by lysis of the cells by three cycles of freezing and thawing. The spontaneous release of histamine–tryptase was estimated from tubes to which buffer, instead of releasing agent, was added. Also, if the baseline release of histamine was greater than 15% of the total histamine release, the cells were deemed damaged during the preparation process and data were excluded. Histamine–tryptase release was calculated as percentage of total release. Net percentage of histamine–tryptase release is expressed as a percentage of total histamine–tryptase corrected for spontaneous release, according to the formula
Net %release = (T − B)/T · 100
where T is total histamine and B is spontaneous release.
Histamine concentrations were measured by the enzyme immunoassay method using commercially available kits (Immunotech, Westbrook, ME). Tryptase concentrations were measured using the UniCap 100 automated apparatus (Pharmacia and Upjohn, Kalamazoo, MI) and the UniCap 100 fluoroenzyme immunoassay.
Mechanisms for Nonimmunologic Degranulation
For elucidating the mechanisms of histamine and tryptase release, two different types of enzymatic inhibitors were studied using a stimulus-secretion paradigm, with histamine and tryptase release from the mast cells as the endpoint.
Phospholipase A2Inhibition by 3-(4-Octadecyl)-benzoylacrylic Acid.
3-(4-Octadecyl)-benzoylacrylic acid (OBAA; BIOMOL Research Laboratories Inc.; Plymouth Meeting, PA) is a potent inhibitor of PLA2. Ten milligrams OBAA was dissolved in 9.34 ml absolute ethanol and split into 500-μl aliquots that were stored at −70°C. Just before use, 40 μl ethanol solution was further diluted with 1:1 solution (vol/vol) of ethanol–PAG to a final concentration of 24 μM. For the inhibition studies, 5-μl aliquots of the ethanol–PAG solution were added to 100 μl cell suspensions (resulting in a final concentration of 1 μM of the inhibitor). Tubes were preincubated for 30 min at 37°C before addition of vancomycin or calcium ionophore A23187, and the cell suspensions were incubated for an additional 30 min. Ethanol in PAG (2.08%) and OBAA without addition of drugs were used as controls.
Phospholipase C Inhibition by (1-(6-((17β-3Methoxyestra-1,3,5(10)-trien-17-yl)amino)hexyl)-1H-pyrrole-2,5-dione (U-73122). (1-(6-((17B-3-Methoxyestra-1,3,5(10)-trien-17-yl)amino)hexyl)-1H-pyrrole-2,5-dione (U-73122; BIOMOL Research Laboratories Inc.) inhibits receptor-coupled generation of inositol 1,4,5-triphosphate and intracellular mobilization of Ca2+(IC50[concentration of the inhibitor that causes 50% of inhibition]= 1.0–2.1 μM) and exhibits little or no direct inhibition of PLA2and phospholipase D.
Five milligrams U-73122 was dissolved in 5 ml methylene chloride and split into 100-μl aliquots, which were stored at −70°C. Just before use, methylene chloride was evaporated under nitrogen and U-73122 was redissolved in 150 μl absolute ethanol. This was further diluted with 927 μl PAG buffer, resulting in a 199.8-μM U-73122 solution. For the inhibition studies, 5 μl PAG–ethanol solution was added to 100 μl cell suspension, resulting in 8.36 μM U-73122. Tubes were preincubated for 30 min at 37°C before addition of vancomycin or calcium ionophore A23187, and the suspensions were incubated for an additional 30 min. Ethanol in PAG (0.58%) and U-73122 without addition of mediators were used as controls.
Results are presented as the mean and SD, and the number of experiments (n) is indicated. All two-group comparisons were performed using a paired or unpaired t test, with the level of statistical significance set at P < 0.05. Multigroup comparisons were made using one-way analysis of variance, followed by a Scheffépost hoc test for multiple comparisons, with P < 0.05 considered to be significant.
Each foreskin yielded between 2 × 105and 17 × 106cells, with 4–8% being mast cells. In the presence of Ca2+and Mg2+in the incubating buffer, we found that vancomycin, calcium ionophore A23187, morphine, and atracurium resulted in significant histamine release (31 ± 5.1, 60 ± 14.2, 17.4 ± 10.2, and 8.8 ± 5.2%, respectively), with an average baseline release of 6.9 ± 3.6% (fig. 1). Tryptase release was 22.7 ± 6.8, 18.1 ± 10.5, 22.2 ± 13.0, and 11.6 ± 6.3, respectively, with an average baseline release of 10.8 ± 5.9% (P < 0.005;fig. 2). Also, there was a direct correlation between the net percentage releases of histamine and tryptase by vancomycin, morphine, and atracurium (r2= 0.6;fig. 3), but no correlation between net percentage releases of histamine and tryptase for calcium ionophore A23287.
If calcium and magnesium were omitted from the incubation buffer, histamine release from neonatal skin mast cells was decreased by 90% for vancomycin (8.5 ± 5.9%; baseline 6.0 ± 4.0%) and virtually abolished for morphine (5.7 ± 2.5%; baseline 5.6 ± 3.5%;P < 0.05). Conversely, calcium had no effect on release reaction of A23187 (55.8 ± 15.1%; baseline 6.5 ± 3.7%;P < 0.0001) or atracurium (5.4 ± 2.5%; baseline 3.7 ± 2.0%;P = 0.0004;fig. 4). Furthermore, there was a significant difference in net percentage histamine release between Ca–Mg(+) and Ca–Mg(−) groups if vancomycin (P < 0.0001) or morphine (P = 0.0012) was used as the mast cell activator, but no difference was seen with calcium ionophore A23187 (P = 0.57) or atracurium (P = 0.85;fig. 5).
Preincubation of the dispersed mast cells with the secretory PLA2inhibitor OBAA attenuated vancomycin-induced histamine release from 19.4 ± 8.5% to 9.3 ± 6.3% (baseline: 6.0 ± 2.7%; control: 5.8 ± 2.3%; OBAA: 6.4 ± 2.9%); whereas the calcium ionophore A23187–induced histamine release was not affected (77.0 ± 28.7 vs. 73.3 ± 17.4%; baseline 7.3 ± 2.0%; control: 7.1 ± 1.1%; OBAA: 7.6 ± 1.6%;fig. 6).
For the PLC inhibitor U-73122 we found a similar pattern as for the PLA2inhibitor: It abolished vancomycin-induced histamine release but not calcium ionophore A23187–induced histamine release. For the vancomycin group, data were as follows: baseline: 5.2 ± 3.4%; control: 5.5 ± 2.8%; U-73122: 8.9 ± 3.8%; U-73122 with vancomycin: 9.3 ± 3.8%; and vancomycin: 35.3 ± 18.7%. For the calcium ionophore A23187 group, values were as follows: baseline: 7.3 ± 3.0%; control: 5.7 ± 2.3%; U-73122: 8.2 ± 3.4%; U73122 and A23187: 80.4 ± 18.9%; and A23187: 77.3 ± 19.4%. The groups were compared using analysis of variance, and we found a significant release for vancomycin alone, for U73122 with A23187, and for A23187 (P = 0.05), compared with the baseline and the control (fig. 7).
We found that tryptase is released in addition to histamine during nonimmunologic processes by known mast cell degranulators. Also, vancomycin, a prototypical mast cell degranulator, appears to be partially dependent on calcium and releases histamine and tryptase via PLC- and PLA2-dependent mechanisms. This may be a result of either direct action on PLC and PLA2by vancomycin or stimulation of an unknown receptor mechanism. Our finding of significant tryptase release by vancomycin differs from the result described in a recent publication by Renz et al. 18Those authors looked at plasma levels of tryptase and histamine in patients receiving rapid vancomycin infusion and saw increases in plasma histamine levels only. The difference in the results may be caused by either direct activation of mast cells in vitro or the fact that only in vivo basophil activation occurred. Conversely, mast cell degranulation induced by calcium ionophore A23187, which causes Ca2+influx and releases Ca2+from intracellular sites, such as the sarcoplasmic reticulum, 19,20was not inhibited by PLC and PLA2inhibitors or decreased in the absence of extracellular calcium. This extracellular calcium independence on the histamine release by A23187 is opposite to the response seen in the dispersed mast cells from human lungs. Church et al. 21reported that histamine release induced by the calcium ionophore A23187 was decreased by more than 90% if calcium was omitted from the incubation medium. Because mast cells are functionally heterogeneous and possess different characteristics, 7,22previously described pathways from animal experiments and nonconnective tissue mast cells may not be valid for human cutaneous infant mast cells, the cells used in this study.
Our mast cell degranulation results are in accordance with other studies. Levy et al. 3showed that vancomycin induced 22.9% histamine release. Benyon et al. 23showed that calcium ionophore A23187 released 28.6% histamine. Stellato et al. 5showed a significant positive correlation between the percentages of histamine and tryptase release induced by morphine from human skin mast cells, and the release was calcium dependent, and, as in our study, no release was observed if calcium was removed from the incubation buffer. 13Atracurium is a known histamine releaser, both in vivo 24and in vitro (net histamine release 12% with 10−3M), 6although another study reports that atracurium causes modest histamine release. 25We also found only a modest release of histamine and tryptase after incubation with atracurium. Tryptase has been shown to be released with histamine from human heart and synovial mast cells, if stimulated immunologically in vitro , 26,27and others have shown that it is released with histamine after an immunoglobulin (Ig) E–mediated event in vivo . 28,29Stellato et al. 5showed a positive correlation between the percentage of histamine and tryptase release induced by buprenorphine or by anti-IgE from human lung mast cells. Watkins 30previously defined the nonimmune mechanisms as absence of plasma tryptase but presence of increased urinary methylhistamine excretion. According to a study by Fisher and Baldo, 31increased concentrations of mast cell tryptase are not associated invariably with immunologic reactions. In a recent study, Edston and van Hage-Hamsten 32found elevated levels of tryptase in heroin addicts who died suddenly after injection, indicating a possible mast cell degranulation. There was no correlation between total IgE and tryptase levels, suggesting that heroin and morphine may have activated mast cells nonimmunologically. 32Data from our own in vitro study shows that tryptase also can be released, along with histamine, by nonimmunologic mast cell activation.
There was marked variability in histamine release from different foreskin preparations (all prepared using the same cell-dispersion protocol), suggesting that the ability to release influences the effect of the drugs on cutaneous mast cells. Furthermore, the number of mast cells isolated per gram of skin varied markedly, which could be another factor that influences drug sensitivity or other nonimmunologic mast cell activators.
The mechanisms for nonimmunologic mast cell activation are not well-described but suggest a dose-dependent and reproducible noncytotoxic reaction. 33Some authors describe the reaction in vitro to be independent of extracellular calcium 1; others describe it as partially calcium-independent 23,34; and still others find it to be dependent on extracellular calcium, with maximum release if incubated with 3-mM extracellular calcium. 4,35–36,37Our studies with neonatal, partially purified, mast cells showed that the release was partially dependent on extracellular calcium, except in the case of the calcium ionophore A23187, which can release calcium from intracellular stores. 19,20
Studies with peritoneal rat mast cells show that PLA2inhibitors can suppress histamine release but not prostaglandin release, if challenged with IgE or nonimmunochemical stimuli, such as calcium ionophore A23187, concanavalin A, compound 48/80, and substance P. 38Murakami et al. 39also showed that if rat mast cells are exposed to type II PLA2, a significant release of histamine is observed. Our studies with the human cutaneous mast cell showed decreased histamine release if PLA2or PLC was inhibited (figs. 6 and 7). Elucidating pathways responsible for the histamine and tryptase release requires further studies to obtain a better understanding of in vivo and in vitro mast cell activation.
Laroche et al. 40measured concentrations of plasma histamine and tryptase after exposure to radiocontrast media in 20 patients with mild to severe reactions. Increased plasma histamine levels developed in 14 patients and increased tryptase levels developed in 16. 40Histamine and tryptase concentrations correlated with the severity of the reaction. The authors also reported the presence of IgE against the radiocontrast agents, although radiocontrast reactions traditionally have been considered to be nonimmunologic anaphylactoid reactions. Tests to determine IgE antibodies against radiocontrast agents are not validated; the authors did not perform inhibition studies to further validate their assay; and radiocontrast reactions occurred without previous exposure, suggesting that the reactions they noted were anaphylactoid.
Based on our in vitro study, we conclude that tryptase is released with histamine during nonimmunologic mast cell activation and also can be an indicator for anaphylactoid reactions because it is only present in the cytoplasmic granules of mast cells. 41The mechanism of nonimmunologic histamine and tryptase release relies on membrane associated enzymes, such as PLA2and PLC. Although the physiologic role of tryptase is not clear, it is a neutral protease that can both activate and inactivate multiple inflammatory mediators. Further elucidation of direct molecular effects of mast cell activation may be important in understanding mechanisms of anaphylactoid reactions and other pathologic states of mast cell activation.
The authors thank Dr. Maria E. Arias and the staff at Georgia Baptist Hospital, Atlanta, Georgia, and Dr. Raphael Gershon and the staff at Grady Memorial Hospital, Atlanta, Georgia, for collecting the foreskins.