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Potentiation of antigen-induced histamine release from rat peritoneal mast cells through a direct interaction between mast cells and non-mast cells
Life Sciences, Vol. 54, No. 19, pp. 1403-1409, 1994 Copyright © 1994 Elsevier Science Ltd Printed in the USA. All rights reserved 0024-3205/94 $6.OO + .OO
Pergamon
P O T E N T I A T I O N OF A N T I G E N - I N D U C E D H I S T A M I N E R E L E A S E F R O M RAT P E R I T O N E A L MAST C E L L S T H R O U G H A D I R E C T I N T E R A C T I O N B E T W E E N MAST C E L L S AND N O N - M A S T C E L L S Naoki Inagaki, Hirokazu Kawasaki, Makoto Ueno, Hiroichi Nagai and Akihide Koda Department of Pharmacology, Gifu Pharmaceutical University 5-6-1, Mitahorahigashi, Gifu 502, Japan (Received in final form February 17, 1994) Summary Rat peritoneal mast cells, which had been sensitized two days earlier by an intraperitoneal injection of rat monoclonal IgE antibodies, were purified by density gradient centrifugation with 60 % Percoll (cell purity >95 %). Histamine release from the purified mast cells (PMC) was then compared to that of a non-purified preparation (peritoneal exudate cells; PEC). Both PEC and PMC released similar amounts of histamine upon stimulation with calcium ionophore A23187 and compound 48/80. In contrast, antigen-induced histamine release from PMC was very low compared to that of PEC. PEC released up to 30 % of total histamine upon challenge with 1 ~tg/ml of antigen, whereas histamine release from PMC was only one third or less than that of PEC. When PEC was suspended in 60 % Percoll and treated for a period needed for purification, the reduction of antigen-induced histamine release was negligible. Mast cells purified by centrifugation on a metrizamide gradient released only small amount of histamine similar to Percollpurified mast cells. Non-mast cells (NMC) recovered from the interface of the 60 % Percoll potentiated the antigen-induced histamine release from PMC concentrationand time-dependently. The supernatant of the NMC suspension which was incubated at 37 °C for 60 min, however, failed to potentiate histamine release in PMC. We concluded therefore that separation media such as Percoll and metrizamide do not cause the low antigen-induced histamine release in PMC, but that the separation of mast cells from other cells present in the peritoneal cavity itself causes it. Antigeninduced mast cell histamine release is potentiated through a direct interaction between mast cells and NMC, and some cell surface molecules also seem to be involved.
Key Words: peritoneal mast cell, histamine release, non-mast cell
The mast cell is an important target cell in the IgE antibody-mediated allergic reaction and releases a variety of mediators upon challenge with specific antigens (1-3). Many humoral factors are reported to cause or modulate mediator release from mast cells, and the reaction is also caused by a variety of non-immunological stimuli (4-8). The peritoneal mast cells of rats, which are obtained as single cells, have been widely used for studying the allergic mediator release (7,9-11), and the cells are easily separated by density gradient centrifugation (6,10-13). We noticed in our preliminary experiments that rat peritoneal mast ceils purified by density gradient centrifugation with Percoll released only a small amount of histamine upon challenge with antigen in comparison with nonpurified preparations (14). Although other investigators have recognized similar problems, the mechanism involved has never been explained. In this report, we offer an explanation for the reduction of antigen-induced histamine release in purified rat peritoneal mast cells.
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Materials and Methods AnimUs. Male Wistar rats weighing 250-300 g were used throughout. Rats were purchased from Japan SLC, Inc. (Hamamatsu, Japan). b4onoclonal leE antibody, antigen, buffer and reagents. Rat anti-dinitrophenol (DNP) monoclonal IgE antibt)dy producing cell line, REC, establisfied in Kissei Pharmaceutical Co., Ltd. (Hotaka, Japan), is maintained in our laboratory. The culture supernatant of REC was used as a source of IgE antibody. The IgE antibody titer of the preparation estimated by homologous passive cutaneous anaphylaxis was 1:2,000 or greater. DNP-conjugated bovine serum albumin (DNP-BSA) prepared according to the method described by Eisen et al. (15) with some modifications was used as an antigen to elicit mast cell histamine release. The number of DNP residues introduced was 25 per BSA molecule. Tyrode solution containing HEPES and gelatin (137 mM NaC1, 2.7 mM KC1, 0.41 mM NaH2PO4, 1.6 mM CaCI2, 1 mM MgC12, 0 . 1 % glucose, 10 mM HEPES, 0.05 % gelatin, pH 7.4) was used throughout. Calcium ionophore A23187 (A23187) and compound 48/80 (48/80) were obtained from Sigma Chemical Co., St. Louis, USA. Purification of mast cells. Peritoneal mast cells were sensitized by an intraperitoneal injection of 2 ml of a 1/200 dilution of monoclonal IgE antibody preparation. Two days later, rats were sacrificed under ether anesthesia, and peritoneal cells were recovered by washing the cavity with 20 ml of Tyrode solution containing 5 units/ml of heparin. Peritoneal washings were pooled and spun down at 45xg for 5 min. Cells were washed 2 times with Tyrode solution by centrifugation at 45xg for 5 min and resuspended in small volume of Tyrode solution. The washed peritoneal cell suspension was divided into 2 or 3 parts as needed. One was served for experiments on non-purified mast cells (peritoneal exudate cells; PEC), which was diluted with Tyrode solution and kept during mast cell purification. Mast cells were purified by density gradient centrifugation with Percoll (Pharmacia LKB Biotechnology, Uppsala, Sweden) (10,13). Ninety per cent isoosmotic Percoll was made by mixing 9 volumes of Percoll with 1 volume of a 10-fold concentration of phosphate-free Tyrode solution, and further diluted with Tyrode solution to make 60 % Percoll, the density of which was 1.09. Into a plastic tube (Falcon #2018), 12 ml of the 60 % Percoll was placed, and 1 ml of the washed peritoneal cell suspension was layered over the Percoll layer. After centrifugation at 45xg for 15 min, cells at the bottom of the tube were collected and suspended in Tyrode solution. In some experiments, non-mast cells (NMC) were also recovered from the interface between the Tyrode solution and 60 % Percoll. Recovered NMC was suspended in about 100 ml of Tyrode solution and centrifuged at 150xg for 10 min. NMC thus obtained were suspended in Tyrode solution. In one experiment, mast cells were also purified by density gradient centrifugation with melrizamide (Sigma) (11,16). One and a half milliliters of 22.5 % of metrizamide in Tyrode solution was placed in a plastic tube (Falcon #2053), and 3 ml of the washed peritoneal cell suspension was layered over the metrizamide layer. After centrifugation at 45xg for 15 min, cells at the bottom of the tube were collected and suspended in Tyrode solution. Suspensions of PEC, purified mast cells (PMC) and NMC were then spun down at 45xg for 5 min, and further washed 2 times with Tyrode solution by centrifugation at 45xg for 5 min. Finally, cells were suspended in appropriate volumes of Tyrode solution, and used as PEC, PMC and NMC. All procedures were performed at room temperature. Mast cells were counted after staining with neutral red. It was shown that all cells that were stained with neutral red could also be stained by toluidin blue, pH 2.5. Viability of cells was 97 % or more assessed by staining with trypan blue. Mast cell purity of PEC and PMC (Percoll-purified) preparations was 5-20 % and over 95 %, respectively. The relatively high percentage of mast cells in PEC preparations may be accounted for by the repeated cell washings by the 45xg, 5 min centrifugation. NMC preparations contained mast cells by O.5-1.5 %. Concentration of mast cells in the final reaction mixture was not fixed. It contained 1-3xllY mast cells/ml.
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Incubation suDernatant of NMC. NMC suspension prepared as mentioned above were divided into 3 equal parts. One part of the suspension was incubated at 37 °C for 60 min and another was placed in an ice-cold bath for the same period. After the 60 min incubation period, the NMC suspension was centrifuged at 350xg for 10 rain and the supernatant was separated. The third portion of the NMC suspension was kept at room temperature, centrifuged at 45xg for 5 min to remove any humoral factors and resuspended in its original volume of Tyrode solution just before use as NMC. Histamine release and assav. An aliquot of the mast cell suspension was placed in a plastic tube (Falcon #2053), and incubated in the absence or presence of NMC, or the incubation supernatant of NMC at 37 °C for specified period of time. Mast cells were then stimulated by adding 50 Ixl of stimulant antigen, A23187 or 48/80, and incubated at 37 °C for 15 min. The final volume of the reaction mixture was 500 tal. To assess the amount of total histamine, cell associated histamine was extracted in the presence of 1.2 % HCIO4. After centrifugation at 350xg for 10 min, the supernatant was separated and stored at -20 °C. The released histamine was measured fluorometrically (17) by a post-column derivatization method on an automated histamine analyzing system (Tosoh Co., Ltd., Tokyo, Japan) (18,19). Samples were centrifuged and filtered to remove precipitates and injected in a volume of 20 ttl. Histamine was separated by an ion exchange column (TSKgel SP-2SW, 150 x 4.6 mm, Tosoh) using a mobile phase of 0.25 M KH2PO4 (0.65 ml/min). The separated histamine was mixed with 3.5 M NaOH (0.1 ml/min) and 0.1% o-phthalaldehyde (0.15 ml/min) in a piece of teflon tubing and coupled with o-phthalaldehyde at 45 °C. The reaction was terminated by mixing with 4 M H3PO4 (0.12 ml/min) and fluorescence intensity was determined. The retention time was 11 min. Mast cell histamine content was 20-40 I.tg/million cells, and spontaneous release of histamine was lower than 3 % of total histamine. In most experiments, the histamine content decreased slightly after purification. Experiments were performed in duplicate, and results were expressed as the mean value of net histamine release. To confirm the results, experiments of the same or similar design were repeated at least two times. Results and Discussion 100
100
20 O
opec 8O
00
0o
6O
4o
4O
O PEC
PEC
O ~
o
| | E
!!•
10
20
z
0.01
0.1
Concentration
1
10 of ,423187 (IJM)
0
0.1
1
10
Con~ntrjlon of 4e~eo(~oJml)
1 I O i~ v w 0.001 0.01 0.1 1 10 100 Concentrstlonof DNP-BSA(l~l/ml)
FIG. 1 Comparison of the histamine release from non-purified mast cells (PFC) and purified mast cells (PMC) caused by calcium ionophore A23187 (A23187), compound 48/80 (48/80) or antigen (DNP-BSA). The peritoneal cell suspension obtained from sensitized rats were divided into 2 parts, one for PFC and the other for purification of mast cells with 60 % Percoll. Mast cell purities of PEC and PMC preparations for experiments of A23187, 48/80 and antigen were 14.4 and 98.5 %, 19.8 and 99.6 %, and 5.4 and 96.8 %, respectively. PEC and PMC suspensions warmed at 37 °(3 for 10 min were incubated in the presence of various concentrations of stimulant at 37 °C for 15 min.
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First, we compared the histamine release from PEC and PMC caused by A23187, 48/80 or antigen. Results are shown in Fig. 1. A23187 released histamine from both PEC and PMC at concentrations of 0.1-10 IxM, and the dose-response curves were quite similar. 48/80 also released histamine from both PEC and PMC at concentrations of 0.3-10 ktg/ml. In contrast to these secretagogues, antigen-induced histamine release from PMC was clearly low compared to that from PEC. The histamine release from PMC was about one half or less than that from PEC after challenge with 0.1-100 Ilg/ml of antigen. The purification-associated reduction of histamine release seems to occur only in the case of immunological stimulus. Antigen-induced histamine release in the following experiments was caused by a submaximum concentration of 1 ~tg/ml of the antigen, which may be an advantageous condition for assessing the effect of a treatment on the reaction. TABLE I Effect of PercolI-Treatment and PercolI-Purification on Antigen-Induced Mast Cell Histamine Release.
PEC
Mast cell purity (%)
Histan~lne content (~g/lO~ mast cells)
Histamine release (% of total)
13.7
34.8
12.1
PEC
Percoll-treated
16.5
32.6
10.7
PMC
Percoll-purlfled
98.4
39.6
1.6
Peritoneal cell suspension obtained from sensitized rats was divided into 3 pads. One was for non-purified mast cells (PEC) and another for purification of mast cells with Percoll (PMC). Cells of the last part were suspended in and treated with 60 % Percoll for a period needed for purification of mast cells of the second part. PEC, Percoll-treated PEC and PMC suspensions warmed at 37 °C for 10 rain were incubated in the presence of 1 IJg/ml of antigen at 37 °C for 15 min. TABLE II Antigen-Induced Mast Cell Histamine Release after Purification with Percoll and Metrizamide.
PEC
Mast cell purity (%)
Hlstan'jine content (V.O/10" mast cells)
Histamine release (% of total)
7.8
32.8
13.3
PMC
Percol I-purified
97.2
27.3
4.0
PMC
Metrlzamide-purlfled
93.6
30.1
2.2
Peritoneal cell suspension obtained from sensitized rats was divided into 3 parts. One was for non-purified mast cells (PEC) and the others for mast cell purification with Percoll and metrizamide (PMC). PEC and PMC suspensions warmed at 37 °C for 10 min were incubated in the presence of 1 IJg/ml of antigen at 37 °C for 15 min.
Next, we examined the effect of purification of mast cells with Percoll on antigen-induced histamine release. As shown in Table 1, PEC released 12.1% of total histamine upon challenge with antigen, whereas PMC released only 1.6 % of total histamine. After treatment with 60 % Percoll, however, the treated PEC released 10.7 % histamine, which was comparable to the amount released by untreated PEC. The slightly lower rate of histamine release in treated PEC may be related to its somewhat higher mast cell purity. Furthermore, as shown in Table 2, PMC separated not only with Percoll but also with metrizamide, released only small percentage of histamine compared to PEC.
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These results indicate that contact with Percoll does not result in the low histamine release in PMC, and that the lowered histamine release in PMC is not a phenomenon specific to PercoU. It seems, therefore, that the separation of mast cells from NMC is crucial, and that the IgE antibody fixed on the mast cell surface is not detached during mast cell purification. TABLE III Restoration of Antigen-Induced Histamine Release from Purified Mast Cells by Reconstitution with Non-Mast Cells. Addition of NMC
Mast cell purity (%)
Histan~lne content (~g/10" mast cells)
PEC
14.7
32.1
13.5
PMC
97.6
26.7
2.5
(prior to challenge)
Histamine release (% of total)
P M C + NMC
20 mln
10.3
24.7
25.6
P M C + NMC
10 mln
10.3
24.7
13.7
P M C + NMC
5 rain
10.3
24.7
11.2
P M C + NMC
0 mln
10.3
24.7
8.8
PMC + NMC
- 5 mln
10.3
24.7
3.2
Peritoneal cell suspension obtained from sensitized rats was divided into 2 parts, one for non-purified mast cells (PEC) and the other for separation of purified mast cells (PMC) and non-mast cells (NMC) with Percoll. NMC suspension contained mast cells at 0.8 %. PEC and PMC suspensions warmed at 37 °C for 20 min were incubated in the presence of 1 IJg/ml of antigen at 37 °C for 15 min. The NMC suspension in a volume of 50 Ill was added to PMC suspension at varying time before or after the antigen stimulation. TABLE IV Restoration of Antigen-Induced Histamine Release from Purified Mast Cells by Reconstitution with Varying Concentration of Non-Mast Cells. Mast cell purity (%)
Hlstan'jlne content (~g/10" mast cells)
Histamine release (% of total)
PEC
12.2
27.8
28.9
PMC
97.6
24.2
7.2
PMC + NMC
1.6 Ill
92.8
23.8
6.4
P M C + NMC
8 Ill
77.6
25.6
9.6
PMC + NMC 40 Id
42.9
26.8
12.5
PMC + NMC 200 ~1
13.5
26.0
20.8
Peritoneal cell suspension obtained from sensitized rats was divided into 2 parts, one for non-purified mast cells (PEC) and the other for separation of purified mast cells (PMC) and non-mast cells (NMC) with Percoll. NMC suspension contained mast cells at 0.5 %. PEC and PMC suspensions warmed at 37 °C for 10 min were incubated in the presence of 1 pg/ml of antigen at 37 °C for 15 min. The NMC suspension was added to PMC suspension at the beginning of preincubation.
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To confmn the role of NMC on mast cell histamine release, the effect of reconstitution of PMC with NMC on histamine release was examined. The results of a time-course study are shown in Table 3. Histamine release from PEC was 13.5 %, whereas that from PMC was only 2.5 %. NMC suspension was added to PMC from 20 min before to 5 min after the antigen stimulation. The mast cell purity after the reconstitution was lowered from 97.6 % to 10.3 %, which was comparable to that of PEC, 14.7 %. The histamine release from PMC was restored by the time-dependent reconstitution with NMC. When NMC was added to PMC 10 min before antigen stimulation, the histamine release reached a level equal to that of PEC. It is interesting that 20 min incubation of PMC with NMC resulted in the histamine release of 25.6 %, which far exceeded the value of PEC, and that addition of NMC with antigen resulted in the partial recovery of histamine release. It seems, therefore, that the condition of and the number of cells in the NMC is different from that of non-mast cells in PEC. Although NMC potentiates some release of histamine even without preincubation, preincubation of PMC with NMC clearly increased the potentiating effect, Addition of NMC 5 min after the antigen stimulation had no effect. The results of the dose-response study are shown in Table 4. PMC was incubated with 1.6-200 ~tl of NMC suspension for 10 min before antigen stimulation. After the reconstitution of PMC with NMC the mast cell purities were in the range of 92.8-13.5 %. The purity of 13.5 % was comparable to that of PEC. Histamine release from PEC and PMC was 28.9 and 7.2 %, respectively. The low histamine release of PMC was recovered by the reconstitution with NMC dose-dependently. These results clearly demonstrate that NMC interacts with mast cells to potentiate antigen-induced histamine release from these cells. TABLE V Failure of Restoration of Antigen-Induced Histamine Release from Purified Mast Cells by Incubation Supernatant of Non-Mast Cells. Mast cell purity (%)
Hlstan~ine content (l~g/10" mast cells)
PEC
9.4
26.2
24.2
PMC
95.7
28.5
7.0
5.7
31.3
34.8
PMC + 0 °C Supernatant
95.7
29.2
9.2
PMC + 37°C Supernatant
95.7
29.7
8.3
PMC + NMC
Histamine release (% of total)
Peritoneal cell suspension obtained from sensitized rats was divided into 2 parts, one for non-purified mast cells (PEC) and the other for separation of purified mast cells (PMC) and non-mast cells (NMC) with Percoll. NMC suspension, which contained mast cells at 1.5 %, was divided into 3 equal parts. One was used as NMC suspension and the others for preparing incubation supematants at 0 °C and 37 °C. PEC and PMC suspensions warmed at 37 °C for 10 min were incubated in the presence of 1 I~g/ml of antigen at 37 °C for 15 min. The final NMC suspension and incubation supematants of NMC in a volume of 200 Id were added to PMC suspension at the beginning of preincubation.
Next, we tried to determine whether humoral factors released from NMC spontaneously are involved in the potentiation of histamine release from PMC. Since the entire experimental process took place during a period of 60 min, we examined the effect of 60 min incubation supernatant of NMC on PMC histamine release. As shown in Table 5, incubation supernatants prepared at 0 °C and at 37 °C failed to potentiate the histamine release from PMC. In the present study, we showed evidence that antigen-induced histamine release from rat peritoneal mast cells is modulated by non-mast cells present in the rat peritoneal cavity. Since our preliminary experiments had shown that a longer incubation period potentiates the release of histamine from PEC, subsequent experiments were designed to be completed in the shortest possible time. Cell counting, therefore, was performed after completion of the histamine releasing reaction.
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As a result, PEC and PMC preparations employed here contained varying concentrations of mast cells (1-3x105 mast cells/ml). We have demonstrated, however, that our results are highly reproducible and consistent. Although many humoral factors which modulate mediator release from mast cells or basophils have been reported (4,7,8,20-21), we indicated that NMC do not secrete effective humoral factors spontaneously. Furthermore, the potentiating effect of NMC on PMC histamine release increased dependently upon the preincubation period of PMC with NMC, indicating that NMC does not release effective molecules after challenge with antigen. We concluded therefore that the direct contact between mast cells and non-mast cells is crucial. In 1989, Thompson et al. indicated that mast cells possess the laminin receptor on their surface, and that they adhere and spread when in contact with laminin (22). Recently, it has been reported that rat peritoneal mast cells stimulated with phorbor ester express VLA-4 and that the signals mediated through VLA-4 potentiate the antigen-induced mediator release from the cells (23). These reports may support our conclusion that antigen-induced rat peritoneal mast cell activation is modulated by direct contact with other cells via some cell surface molecule(s). R~ferences 1. 2. 3. 4.
5. 6. 7.
8. 9,
10. 11. 12. 13. 14. 15. 16. 17. 18. 19. 20. 21. 22.
23.
K. ISHIZAKA and T. ISHIZAKA, Allergy. Princioles and Practice. E. Middleton, Jr., C.E. Reed and E.F. Ellis (eds), 43-73, CV Mosby Company, St. Louis (1983). M.K. CHURCH and J.P. CAUFIELD, Allergy, S.T. Holgate and M.K. Church (eds), 5.1-5.12, Gower Medical Publishing, London (1993). J. BROSTOFF and T. HALL, Immunoloov, I. Roift, J. Brostoff and D. Male (eds), 19.1-19.22, Mosby-Year Book Europe, London (1993). M.V. WHITE, Th~ Mast Cell in! Health and Disease. M.A. Kaliner and D.D. Metcalfe (eds), 109-128, Marcel Dekker, New York (1993). D.E. COCHRANE and W.W. DOUGLAS, Proc. Natl. Acad. Sci. USA 7 l 408-412 (1974). Y. KOIBUCHI, A. ICHIKAWA, N. NAKAGAWA and K. TOMITA, Eur. J. Pharmacol. 115 163-170 (1985). M. SWlETER, W.A. GHALI, C. RIMMER and D. BEFUS, Immunology, 66 606-610 (1989). S.C. BISCHOFF and C.A. DAHINDEN, J. Exp. Med. 175 237-244 (1992). T.J. SULLIVAN, K.L. PARKER, A. KULCZYCKI, JR. and C.W. PARKER, J. Immunol. 117 713-716 (1976). E. WELLIS and J. MANN, Blochem. Pharrnacol. 32 837-842 (1983). H. SAITO, K. ISHIZAKA and T. ISHIZAKA, J. Imrnunol. 143 250-258 (1989). T.J. SULLIVAN, K.L. PARKER, W. STENSON and C.W. PARKER, J. Immunol. 114 1473-1479 (1975). L. ENERBACK and I. SVENSSON, J. Immunol. Methods 39 135-145 (1980). H. NISHIDA, H. MATSUI and H. NAGAI, Arch. Toxicol. 66514-517 (1992). H.N. EISEN, S. BELMAN and M.E. CARSTEN, J. Am. Chem. Soc. 75 4583-4585 (1953). L.B. SCHWARTZ, K.F. AUSTEN and S.I. WASSERMAN, J. Immunol. 123 1445-1450 (1979). C.D. MAY, M. LYMAN, R. ALBERTO and J. CHENG, J. Allergy 46 12-20 (1970). A. YAMATODANI, H. FUKUDA, H. WADA and T. IWAEDA, J. Chromatogr. 344 115-123 (1985). N. INAGAKI, T. MIURA, H. NAGAI and A. KODA, Life Sci. 51 PL201-PL205 (1992). K. HIRAI, Y. MORITA, Y. MISAKI, K. OHTA, T. TAKAISHI S. SUZUKI, K. MOTOYOSHI and T. MIYAMOTO, J. Immunol. 141 3958-3964 (1988). Y. KURIMOTO, A.L. DEWECK and C.A. DAHINDEN, J. Exp. Med. 170 467-479 (1989). H.L. THOMPSON, P.D. BURBELO, Y. YAMADA, H.K. KLEINMAN and D.D. METCALFE, J. Immunol. 143 4188-4192 (1989). M. YASUDA, Y. HASUNUMA, H. ADACHI, T. SEKINE, C. RA, H. YAGITA, S. HIROSE and K. OKUMURA, Proc. Jpn. Soc. Immunol. 22 346 (1992).
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P O T E N T I A T I O N OF A N T I G E N - I N D U C E D H I S T A M I N E R E L E A S E F R O M RAT P E R I T O N E A L MAST C E L L S T H R O U G H A D I R E C T I N T E R A C T I O N B E T W E E N MAST C E L L S AND N O N - M A S T C E L L S Naoki Inagaki, Hirokazu Kawasaki, Makoto Ueno, Hiroichi Nagai and Akihide Koda Department of Pharmacology, Gifu Pharmaceutical University 5-6-1, Mitahorahigashi, Gifu 502, Japan (Received in final form February 17, 1994) Summary Rat peritoneal mast cells, which had been sensitized two days earlier by an intraperitoneal injection of rat monoclonal IgE antibodies, were purified by density gradient centrifugation with 60 % Percoll (cell purity >95 %). Histamine release from the purified mast cells (PMC) was then compared to that of a non-purified preparation (peritoneal exudate cells; PEC). Both PEC and PMC released similar amounts of histamine upon stimulation with calcium ionophore A23187 and compound 48/80. In contrast, antigen-induced histamine release from PMC was very low compared to that of PEC. PEC released up to 30 % of total histamine upon challenge with 1 ~tg/ml of antigen, whereas histamine release from PMC was only one third or less than that of PEC. When PEC was suspended in 60 % Percoll and treated for a period needed for purification, the reduction of antigen-induced histamine release was negligible. Mast cells purified by centrifugation on a metrizamide gradient released only small amount of histamine similar to Percollpurified mast cells. Non-mast cells (NMC) recovered from the interface of the 60 % Percoll potentiated the antigen-induced histamine release from PMC concentrationand time-dependently. The supernatant of the NMC suspension which was incubated at 37 °C for 60 min, however, failed to potentiate histamine release in PMC. We concluded therefore that separation media such as Percoll and metrizamide do not cause the low antigen-induced histamine release in PMC, but that the separation of mast cells from other cells present in the peritoneal cavity itself causes it. Antigeninduced mast cell histamine release is potentiated through a direct interaction between mast cells and NMC, and some cell surface molecules also seem to be involved.
Key Words: peritoneal mast cell, histamine release, non-mast cell
The mast cell is an important target cell in the IgE antibody-mediated allergic reaction and releases a variety of mediators upon challenge with specific antigens (1-3). Many humoral factors are reported to cause or modulate mediator release from mast cells, and the reaction is also caused by a variety of non-immunological stimuli (4-8). The peritoneal mast cells of rats, which are obtained as single cells, have been widely used for studying the allergic mediator release (7,9-11), and the cells are easily separated by density gradient centrifugation (6,10-13). We noticed in our preliminary experiments that rat peritoneal mast ceils purified by density gradient centrifugation with Percoll released only a small amount of histamine upon challenge with antigen in comparison with nonpurified preparations (14). Although other investigators have recognized similar problems, the mechanism involved has never been explained. In this report, we offer an explanation for the reduction of antigen-induced histamine release in purified rat peritoneal mast cells.
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Potentiation of Mast Cell Activation
Vol. 54, No. 19, 1994
Materials and Methods AnimUs. Male Wistar rats weighing 250-300 g were used throughout. Rats were purchased from Japan SLC, Inc. (Hamamatsu, Japan). b4onoclonal leE antibody, antigen, buffer and reagents. Rat anti-dinitrophenol (DNP) monoclonal IgE antibt)dy producing cell line, REC, establisfied in Kissei Pharmaceutical Co., Ltd. (Hotaka, Japan), is maintained in our laboratory. The culture supernatant of REC was used as a source of IgE antibody. The IgE antibody titer of the preparation estimated by homologous passive cutaneous anaphylaxis was 1:2,000 or greater. DNP-conjugated bovine serum albumin (DNP-BSA) prepared according to the method described by Eisen et al. (15) with some modifications was used as an antigen to elicit mast cell histamine release. The number of DNP residues introduced was 25 per BSA molecule. Tyrode solution containing HEPES and gelatin (137 mM NaC1, 2.7 mM KC1, 0.41 mM NaH2PO4, 1.6 mM CaCI2, 1 mM MgC12, 0 . 1 % glucose, 10 mM HEPES, 0.05 % gelatin, pH 7.4) was used throughout. Calcium ionophore A23187 (A23187) and compound 48/80 (48/80) were obtained from Sigma Chemical Co., St. Louis, USA. Purification of mast cells. Peritoneal mast cells were sensitized by an intraperitoneal injection of 2 ml of a 1/200 dilution of monoclonal IgE antibody preparation. Two days later, rats were sacrificed under ether anesthesia, and peritoneal cells were recovered by washing the cavity with 20 ml of Tyrode solution containing 5 units/ml of heparin. Peritoneal washings were pooled and spun down at 45xg for 5 min. Cells were washed 2 times with Tyrode solution by centrifugation at 45xg for 5 min and resuspended in small volume of Tyrode solution. The washed peritoneal cell suspension was divided into 2 or 3 parts as needed. One was served for experiments on non-purified mast cells (peritoneal exudate cells; PEC), which was diluted with Tyrode solution and kept during mast cell purification. Mast cells were purified by density gradient centrifugation with Percoll (Pharmacia LKB Biotechnology, Uppsala, Sweden) (10,13). Ninety per cent isoosmotic Percoll was made by mixing 9 volumes of Percoll with 1 volume of a 10-fold concentration of phosphate-free Tyrode solution, and further diluted with Tyrode solution to make 60 % Percoll, the density of which was 1.09. Into a plastic tube (Falcon #2018), 12 ml of the 60 % Percoll was placed, and 1 ml of the washed peritoneal cell suspension was layered over the Percoll layer. After centrifugation at 45xg for 15 min, cells at the bottom of the tube were collected and suspended in Tyrode solution. In some experiments, non-mast cells (NMC) were also recovered from the interface between the Tyrode solution and 60 % Percoll. Recovered NMC was suspended in about 100 ml of Tyrode solution and centrifuged at 150xg for 10 min. NMC thus obtained were suspended in Tyrode solution. In one experiment, mast cells were also purified by density gradient centrifugation with melrizamide (Sigma) (11,16). One and a half milliliters of 22.5 % of metrizamide in Tyrode solution was placed in a plastic tube (Falcon #2053), and 3 ml of the washed peritoneal cell suspension was layered over the metrizamide layer. After centrifugation at 45xg for 15 min, cells at the bottom of the tube were collected and suspended in Tyrode solution. Suspensions of PEC, purified mast cells (PMC) and NMC were then spun down at 45xg for 5 min, and further washed 2 times with Tyrode solution by centrifugation at 45xg for 5 min. Finally, cells were suspended in appropriate volumes of Tyrode solution, and used as PEC, PMC and NMC. All procedures were performed at room temperature. Mast cells were counted after staining with neutral red. It was shown that all cells that were stained with neutral red could also be stained by toluidin blue, pH 2.5. Viability of cells was 97 % or more assessed by staining with trypan blue. Mast cell purity of PEC and PMC (Percoll-purified) preparations was 5-20 % and over 95 %, respectively. The relatively high percentage of mast cells in PEC preparations may be accounted for by the repeated cell washings by the 45xg, 5 min centrifugation. NMC preparations contained mast cells by O.5-1.5 %. Concentration of mast cells in the final reaction mixture was not fixed. It contained 1-3xllY mast cells/ml.
Vol. 54, No. 19, 1994
Potentiation of Mast Cell Activation
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Incubation suDernatant of NMC. NMC suspension prepared as mentioned above were divided into 3 equal parts. One part of the suspension was incubated at 37 °C for 60 min and another was placed in an ice-cold bath for the same period. After the 60 min incubation period, the NMC suspension was centrifuged at 350xg for 10 rain and the supernatant was separated. The third portion of the NMC suspension was kept at room temperature, centrifuged at 45xg for 5 min to remove any humoral factors and resuspended in its original volume of Tyrode solution just before use as NMC. Histamine release and assav. An aliquot of the mast cell suspension was placed in a plastic tube (Falcon #2053), and incubated in the absence or presence of NMC, or the incubation supernatant of NMC at 37 °C for specified period of time. Mast cells were then stimulated by adding 50 Ixl of stimulant antigen, A23187 or 48/80, and incubated at 37 °C for 15 min. The final volume of the reaction mixture was 500 tal. To assess the amount of total histamine, cell associated histamine was extracted in the presence of 1.2 % HCIO4. After centrifugation at 350xg for 10 min, the supernatant was separated and stored at -20 °C. The released histamine was measured fluorometrically (17) by a post-column derivatization method on an automated histamine analyzing system (Tosoh Co., Ltd., Tokyo, Japan) (18,19). Samples were centrifuged and filtered to remove precipitates and injected in a volume of 20 ttl. Histamine was separated by an ion exchange column (TSKgel SP-2SW, 150 x 4.6 mm, Tosoh) using a mobile phase of 0.25 M KH2PO4 (0.65 ml/min). The separated histamine was mixed with 3.5 M NaOH (0.1 ml/min) and 0.1% o-phthalaldehyde (0.15 ml/min) in a piece of teflon tubing and coupled with o-phthalaldehyde at 45 °C. The reaction was terminated by mixing with 4 M H3PO4 (0.12 ml/min) and fluorescence intensity was determined. The retention time was 11 min. Mast cell histamine content was 20-40 I.tg/million cells, and spontaneous release of histamine was lower than 3 % of total histamine. In most experiments, the histamine content decreased slightly after purification. Experiments were performed in duplicate, and results were expressed as the mean value of net histamine release. To confirm the results, experiments of the same or similar design were repeated at least two times. Results and Discussion 100
100
20 O
opec 8O
00
0o
6O
4o
4O
O PEC
PEC
O ~
o
| | E
!!•
10
20
z
0.01
0.1
Concentration
1
10 of ,423187 (IJM)
0
0.1
1
10
Con~ntrjlon of 4e~eo(~oJml)
1 I O i~ v w 0.001 0.01 0.1 1 10 100 Concentrstlonof DNP-BSA(l~l/ml)
FIG. 1 Comparison of the histamine release from non-purified mast cells (PFC) and purified mast cells (PMC) caused by calcium ionophore A23187 (A23187), compound 48/80 (48/80) or antigen (DNP-BSA). The peritoneal cell suspension obtained from sensitized rats were divided into 2 parts, one for PFC and the other for purification of mast cells with 60 % Percoll. Mast cell purities of PEC and PMC preparations for experiments of A23187, 48/80 and antigen were 14.4 and 98.5 %, 19.8 and 99.6 %, and 5.4 and 96.8 %, respectively. PEC and PMC suspensions warmed at 37 °(3 for 10 min were incubated in the presence of various concentrations of stimulant at 37 °C for 15 min.
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Potentiation of Mast Cell Activation
Voi. 54, No. 19, 1994
First, we compared the histamine release from PEC and PMC caused by A23187, 48/80 or antigen. Results are shown in Fig. 1. A23187 released histamine from both PEC and PMC at concentrations of 0.1-10 IxM, and the dose-response curves were quite similar. 48/80 also released histamine from both PEC and PMC at concentrations of 0.3-10 ktg/ml. In contrast to these secretagogues, antigen-induced histamine release from PMC was clearly low compared to that from PEC. The histamine release from PMC was about one half or less than that from PEC after challenge with 0.1-100 Ilg/ml of antigen. The purification-associated reduction of histamine release seems to occur only in the case of immunological stimulus. Antigen-induced histamine release in the following experiments was caused by a submaximum concentration of 1 ~tg/ml of the antigen, which may be an advantageous condition for assessing the effect of a treatment on the reaction. TABLE I Effect of PercolI-Treatment and PercolI-Purification on Antigen-Induced Mast Cell Histamine Release.
PEC
Mast cell purity (%)
Histan~lne content (~g/lO~ mast cells)
Histamine release (% of total)
13.7
34.8
12.1
PEC
Percoll-treated
16.5
32.6
10.7
PMC
Percoll-purlfled
98.4
39.6
1.6
Peritoneal cell suspension obtained from sensitized rats was divided into 3 pads. One was for non-purified mast cells (PEC) and another for purification of mast cells with Percoll (PMC). Cells of the last part were suspended in and treated with 60 % Percoll for a period needed for purification of mast cells of the second part. PEC, Percoll-treated PEC and PMC suspensions warmed at 37 °C for 10 rain were incubated in the presence of 1 IJg/ml of antigen at 37 °C for 15 min. TABLE II Antigen-Induced Mast Cell Histamine Release after Purification with Percoll and Metrizamide.
PEC
Mast cell purity (%)
Hlstan'jine content (V.O/10" mast cells)
Histamine release (% of total)
7.8
32.8
13.3
PMC
Percol I-purified
97.2
27.3
4.0
PMC
Metrlzamide-purlfled
93.6
30.1
2.2
Peritoneal cell suspension obtained from sensitized rats was divided into 3 parts. One was for non-purified mast cells (PEC) and the others for mast cell purification with Percoll and metrizamide (PMC). PEC and PMC suspensions warmed at 37 °C for 10 min were incubated in the presence of 1 IJg/ml of antigen at 37 °C for 15 min.
Next, we examined the effect of purification of mast cells with Percoll on antigen-induced histamine release. As shown in Table 1, PEC released 12.1% of total histamine upon challenge with antigen, whereas PMC released only 1.6 % of total histamine. After treatment with 60 % Percoll, however, the treated PEC released 10.7 % histamine, which was comparable to the amount released by untreated PEC. The slightly lower rate of histamine release in treated PEC may be related to its somewhat higher mast cell purity. Furthermore, as shown in Table 2, PMC separated not only with Percoll but also with metrizamide, released only small percentage of histamine compared to PEC.
Vol. 54, No. 19, 1994
Potentiation of Mast Cell Activation
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These results indicate that contact with Percoll does not result in the low histamine release in PMC, and that the lowered histamine release in PMC is not a phenomenon specific to PercoU. It seems, therefore, that the separation of mast cells from NMC is crucial, and that the IgE antibody fixed on the mast cell surface is not detached during mast cell purification. TABLE III Restoration of Antigen-Induced Histamine Release from Purified Mast Cells by Reconstitution with Non-Mast Cells. Addition of NMC
Mast cell purity (%)
Histan~lne content (~g/10" mast cells)
PEC
14.7
32.1
13.5
PMC
97.6
26.7
2.5
(prior to challenge)
Histamine release (% of total)
P M C + NMC
20 mln
10.3
24.7
25.6
P M C + NMC
10 mln
10.3
24.7
13.7
P M C + NMC
5 rain
10.3
24.7
11.2
P M C + NMC
0 mln
10.3
24.7
8.8
PMC + NMC
- 5 mln
10.3
24.7
3.2
Peritoneal cell suspension obtained from sensitized rats was divided into 2 parts, one for non-purified mast cells (PEC) and the other for separation of purified mast cells (PMC) and non-mast cells (NMC) with Percoll. NMC suspension contained mast cells at 0.8 %. PEC and PMC suspensions warmed at 37 °C for 20 min were incubated in the presence of 1 IJg/ml of antigen at 37 °C for 15 min. The NMC suspension in a volume of 50 Ill was added to PMC suspension at varying time before or after the antigen stimulation. TABLE IV Restoration of Antigen-Induced Histamine Release from Purified Mast Cells by Reconstitution with Varying Concentration of Non-Mast Cells. Mast cell purity (%)
Hlstan'jlne content (~g/10" mast cells)
Histamine release (% of total)
PEC
12.2
27.8
28.9
PMC
97.6
24.2
7.2
PMC + NMC
1.6 Ill
92.8
23.8
6.4
P M C + NMC
8 Ill
77.6
25.6
9.6
PMC + NMC 40 Id
42.9
26.8
12.5
PMC + NMC 200 ~1
13.5
26.0
20.8
Peritoneal cell suspension obtained from sensitized rats was divided into 2 parts, one for non-purified mast cells (PEC) and the other for separation of purified mast cells (PMC) and non-mast cells (NMC) with Percoll. NMC suspension contained mast cells at 0.5 %. PEC and PMC suspensions warmed at 37 °C for 10 min were incubated in the presence of 1 pg/ml of antigen at 37 °C for 15 min. The NMC suspension was added to PMC suspension at the beginning of preincubation.
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Potentiation of Mast Cell Activation
Vol. 54, No. 19, 1994
To confmn the role of NMC on mast cell histamine release, the effect of reconstitution of PMC with NMC on histamine release was examined. The results of a time-course study are shown in Table 3. Histamine release from PEC was 13.5 %, whereas that from PMC was only 2.5 %. NMC suspension was added to PMC from 20 min before to 5 min after the antigen stimulation. The mast cell purity after the reconstitution was lowered from 97.6 % to 10.3 %, which was comparable to that of PEC, 14.7 %. The histamine release from PMC was restored by the time-dependent reconstitution with NMC. When NMC was added to PMC 10 min before antigen stimulation, the histamine release reached a level equal to that of PEC. It is interesting that 20 min incubation of PMC with NMC resulted in the histamine release of 25.6 %, which far exceeded the value of PEC, and that addition of NMC with antigen resulted in the partial recovery of histamine release. It seems, therefore, that the condition of and the number of cells in the NMC is different from that of non-mast cells in PEC. Although NMC potentiates some release of histamine even without preincubation, preincubation of PMC with NMC clearly increased the potentiating effect, Addition of NMC 5 min after the antigen stimulation had no effect. The results of the dose-response study are shown in Table 4. PMC was incubated with 1.6-200 ~tl of NMC suspension for 10 min before antigen stimulation. After the reconstitution of PMC with NMC the mast cell purities were in the range of 92.8-13.5 %. The purity of 13.5 % was comparable to that of PEC. Histamine release from PEC and PMC was 28.9 and 7.2 %, respectively. The low histamine release of PMC was recovered by the reconstitution with NMC dose-dependently. These results clearly demonstrate that NMC interacts with mast cells to potentiate antigen-induced histamine release from these cells. TABLE V Failure of Restoration of Antigen-Induced Histamine Release from Purified Mast Cells by Incubation Supernatant of Non-Mast Cells. Mast cell purity (%)
Hlstan~ine content (l~g/10" mast cells)
PEC
9.4
26.2
24.2
PMC
95.7
28.5
7.0
5.7
31.3
34.8
PMC + 0 °C Supernatant
95.7
29.2
9.2
PMC + 37°C Supernatant
95.7
29.7
8.3
PMC + NMC
Histamine release (% of total)
Peritoneal cell suspension obtained from sensitized rats was divided into 2 parts, one for non-purified mast cells (PEC) and the other for separation of purified mast cells (PMC) and non-mast cells (NMC) with Percoll. NMC suspension, which contained mast cells at 1.5 %, was divided into 3 equal parts. One was used as NMC suspension and the others for preparing incubation supematants at 0 °C and 37 °C. PEC and PMC suspensions warmed at 37 °C for 10 min were incubated in the presence of 1 I~g/ml of antigen at 37 °C for 15 min. The final NMC suspension and incubation supematants of NMC in a volume of 200 Id were added to PMC suspension at the beginning of preincubation.
Next, we tried to determine whether humoral factors released from NMC spontaneously are involved in the potentiation of histamine release from PMC. Since the entire experimental process took place during a period of 60 min, we examined the effect of 60 min incubation supernatant of NMC on PMC histamine release. As shown in Table 5, incubation supernatants prepared at 0 °C and at 37 °C failed to potentiate the histamine release from PMC. In the present study, we showed evidence that antigen-induced histamine release from rat peritoneal mast cells is modulated by non-mast cells present in the rat peritoneal cavity. Since our preliminary experiments had shown that a longer incubation period potentiates the release of histamine from PEC, subsequent experiments were designed to be completed in the shortest possible time. Cell counting, therefore, was performed after completion of the histamine releasing reaction.
Vol. 54, No. 19, 1994
Potentiation of Mast Cell Activation
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As a result, PEC and PMC preparations employed here contained varying concentrations of mast cells (1-3x105 mast cells/ml). We have demonstrated, however, that our results are highly reproducible and consistent. Although many humoral factors which modulate mediator release from mast cells or basophils have been reported (4,7,8,20-21), we indicated that NMC do not secrete effective humoral factors spontaneously. Furthermore, the potentiating effect of NMC on PMC histamine release increased dependently upon the preincubation period of PMC with NMC, indicating that NMC does not release effective molecules after challenge with antigen. We concluded therefore that the direct contact between mast cells and non-mast cells is crucial. In 1989, Thompson et al. indicated that mast cells possess the laminin receptor on their surface, and that they adhere and spread when in contact with laminin (22). Recently, it has been reported that rat peritoneal mast cells stimulated with phorbor ester express VLA-4 and that the signals mediated through VLA-4 potentiate the antigen-induced mediator release from the cells (23). These reports may support our conclusion that antigen-induced rat peritoneal mast cell activation is modulated by direct contact with other cells via some cell surface molecule(s). R~ferences 1. 2. 3. 4.
5. 6. 7.
8. 9,
10. 11. 12. 13. 14. 15. 16. 17. 18. 19. 20. 21. 22.
23.
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