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New role for heparan sulfate: Regulator of leukotriene generation in mouse E-mast cells
Life Sciences, Vol. 39, pp. 903-910 Printed in the U.S.A.
Pergamon Journals
NEW ROLE FOR HEPARAN SULFATE: REGULATOR OF LEUKOTRIENE GENERATION IN MOUSE E-MAST CELLS M. Shalit,*
H. Shoam,**
N. Seno,*** and E. Razin**
*The Department Of Medicine, Hadassah University Hospital, **The Institute for Biochemistry, Hebrew University-Hadassah Medical School Jerusalem,lsrael ***Department of Chemistry, Ochanomizu University, Tokyo, Japan. (Received in final form June i0, 1986) Summary In this work, bovine heparan sulfate, pig mucosa heparin and squid chondroitin sulfate-E glycosaminoglycans (GAGs) were compared as to their affect on the synthesis of leukotrienes C 4 (LTC4) and B 4 (LTB4) as well as on the production of prostaglandin D 2 (PGD2) in cultured mouse E-mast cells (E-MC). The maximum percent increase in LTC 4 generation in cells treated with 0.2 ~g heparan sulfate was 52 ± 3% (mean ± S.E., n = 5). Whereas 0.5 ~g of the GAG increased the production of LTB 4 by 50% Ten micrograms of heparin slightly increased the LTC 4 production (33%) whereas lower doses were found to be ineffective. No significant increase in LTC 4 production was demonstrated when the IgE sensitized E-MC were treated with chondroitin sulfate-E GAG prior to the antigen challenge. Neither one of the three GAGs, at the various doses used, affected the antigen induced exocytosis of ~-hexosaminidase from the IgE sensitized B-MC. After 15 min preincubation with heparan sulfate, antigen induced release of PGD 2 from 1 x 106 sensitized cells was inhibited from 5.4 ng ± 0.I ng into 3.5 ng ± 0.i ng at 0.5 ~ g/ml GAG. All of the three types of GAGs used were uneffective when the E-MC were activated by calcium ionopore A23187. Upon stimulation by IgE-antigen or calcium ionophore A23187, cultured mouse E-~C release preformed mediators and produce oxidative metabolites of arachidonic acid predominantly through the 5-1ipoxygenase pathway (1,2,3). Leukotriene C 4 (LTC4) and B 4 (LTB4) are two of the major components of the 5-1ipoxygenase pathway within the mouse E-MC (1,4). The mechanisms for regulating leukotriene synthesis are not fully understood. This study focused on the role of heparan sulfate and heparin GAGs in the regulation of leukotriene synthesis in cultured mouse E-MC. Heparan sulfate is closely related to heparin GAG. It is characterized by a higher content of N-acetyl groups than is heparin while the content of N-sulfate groups are lower (5). Its predominant uronic acid is glucoronic acid, as opposed to iduronic acid in heparin. The degree of O-sulfation is lower and Correspondence: Dr. Ehud Razin, The Institute of Biochemistry, Hebrew University-Hadassah Medical School, P.O.Box 1172, Jerusalem, Israel. 0024-3205/86 $3.00 + .00 Copyright (c) 1986 Pergamon Journals Ltd.
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corresponds to that of the iduronic acid and N-sulfate content (5). From a biological viewpoint, heparin is characteristically stored in the granule of mast cells, from which it can be released in response to certain stimuli. Heparan sulfate proteoglycan, on the other hand, is an ubiquitous component of cell surfaces of many or all cell types (5). This study demonstrates that heparan and heparin GAG effectively regulate the leukotrienes synthesis after IgE-antigen stimulation of mouse E-MC. However, they were uneffective when the cells were activated by calcium ionopore A23187.
Materials and Methods Materials: Male BALB/C mice (Hebrew University animal breeding center, Jerusalem); WEHI-3 cell line (3); RPMI--1640, L-glutamine, non-essential amino acids, penicillin and streptomycin (Grand Island Biological Co., Grand Island, NY); fetal calf serum (FCS) (Biological Industries, Kibbutz Beit Haemek Israel); B-2-mercaptoethanol (Fisher Scientific, Medford, MA); P--nitrophenyl-B-D-acetamido--2-deoxyglucopyranoside; pig mucosa heparin (Sigma Chemical Co., St. Louis, MO); bovine heparan sulfate purified by chromatography (6) (Seikagaku Kogyo, Tokyo, Japan); squid chondroitin sulfate-E (8), calcium ionophore A23187 (Calbiochem-Behring Corp, La Jolla, Ca); 3H-LTC4 3H-LTB 4, and 3H-prostaglandin D 2 (PGD 2) (New England Nuclear Corp. Boston, MA) synthetic leukotrienes and antibodies against them (kindly provided by Dr. J. Rokach Merck, Canada); synthetic PGD 2 and antibodies against it (kindly provided by Dr. L. Levine, Boston, MA); mouse monoclonal anti-dinitrophenyl IgE (DNP) and DNP coupled to bovine serum albumin (DN-P--BSA) (kindly provided by Dr. F.-T. Liu, Medical Biology Institute, La Jolla, CA). Differentiation and sensitization of mouse E-MC: Bone marrow cells obtained from femurs of 2 month old BALB/C mice were cultured at 37°C in a humidified atmosphere containing 5% CO 2 at a starting density of 0.I x lO 6 cells/ml in RPMI 1640 supplemented with 10% FCS, 2 mM L-glutamine, O.1 mM nonessential amino acids, lO0 U/ml penicillin, lO0 ~g/ml Streptomycin, and 50 ~M B-2-mercaptoethanol at pH 7.2 (enriched medium); The enriched medium was supplemented with 50% WEHI--3 conditioned medium (WEHI-3-CM). WEHI-3-CM was produced by seeding WEHI--3 cells at 1 x 106/ml into the enriched medium and incubating them for 4 days at 37°C in 5% CO 2. Every 7 days the nonadherent cells from the bone marrow cultures were transferred into fresh enriched medium containing 50% WEHI--3-CM. The cultures were maintained for 14 days to obtain a F.-MC population of 97% purity~ as assessed by histological staining (3). Replicate samples of 1 x lOv E-MC were suspended in 0.2 ml of Tyrode's buffer containing 1 mM Ca 2+, 0.2 mM Mg 2+ and 0.05% gelatin (TG) and were sensitized by incubation for 1 hr at 37°C with 5 ~g/lO b cells of mouse monoclonal anti DNP IgE (2,3). Modulation of the activation response: Sensitized cells were washed with 1.5 ml of TG and sedimented at 400 x g at room temperature, they were then suspended in 400 ~l of TG
Vol. 39, No. i0, 1986
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containing a defined concentrations of heparan sulfate or heparin or chondroitin snlfate-E GAGs and were incubated for 15 min at 37°C. 50 ng of DNP--BSA in I00 ~I TG were added to each sample and the incubation was continued at 37°C for I0 more minutes. Unsensitized cells (i x 106 ) in 400 ~i TG containing defined concentrations of each GAG were preincubated for 15 min at 37 ° C and activated for 30 min at 37°C by the addition of i00 ~i TG containing calcium ionophore A23187 final concentration of 0.05 and 0.2 ~M. All experiments were carried out in duplicate and included unchallenged controls for both sensitized and unsensitized cells. Cell viability was greater than 95% at the highest concentration used for each GAG. This was determined by trypan blue exclusion before as well as after antigen or ionophore activation. The antigen and ionophore activated cells were sedlmented at 400 x g for 5 mln at 22°C, and the supernatants were harvested. The cell sediments were resuspended in 500 BI of TG and sonicated at 4°C wi=h a Sonifier. Mediator assays: 8-Hexosaminidase was 2-deoxy-glucoparanoside : I 37°C (3). The results are B-hexosaminidase calculated
assayed by hydrolysis of p-nitrophenyl-B-DU of enzyme cleaves to I- mol of substrate/hr at expressed as the net percentage of release of according to the formula:
net percent release
S-Scontrol (S + P) - Scontrol
in which S = enzyme content of supernatant from stimulated cells. P = enzyme content of sediment from stimulated cells and Scontrol = enzyme content of supernatant from unstimulated cells. Radioimmunoassay (RIA) for the LTC 4 and PGD 2 was performed as described (8). RIA for LTB 4 was performed as described (9). In addition, duplicate samples of 400 ~I supernatants from the activated cells were mixed with 4 vol of methanol for 30 min at 4°C, centrifuged at 1500 x g for 5 min at 4°C to remove precipitated proteins. The extractions were either introduced on to Reverse Phase High Performance Liquid Chromatography (RP-HPLC) to detect leukotrienes, or dried and reconstituted with chloroform:methanol (2:1). Then the samples were loaded on thin layer plates (silica gel G 250 um, Brinkman), and developed in water-saturated ethyl acetate:2,2,4-trimethylpentane:acetic acid (110:50:0.8) together with PGD 2 standard. The PGD 2 fractions were scraped, extracted with methanol, dried and reconstituted with the RIA medium and the amount of PGD 2 was determined by the RIA. The leukotrienes were resolved by RP-HPLC with an isocratic solvent of methanol:water:acetic acid (65:34.9:0.1, pH 5.6) at a flow rate of 1 ml/min. Fractions were evaporated to dryness and redissolved in 500 ~i of the RIA medium for measurement of their leukotriene's immunoreactivity. The various GAGs did not interfere with the various RIA. This was determined after
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Heparin Sulfate and 5-Lipoxygenase
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comparison of the standard curve performed with or without the addition of the GAGs. In order to quantitate the LTC 4 remaining in the pellet of IgE-antigen stimulated cells incubated with or without lO ~g heparin or heparan sulfate, the pellets were mixed with 4 vol of methanol, incubated for 30 min at 4°C and centrifuged at 1500 x g for lO mins at 4°C to remove precipitated proteins. Each supernatant was evaporated to dryness, redissolved in 0.5 ml saline and assayed for LTC 4 by the RIA.
Results and Dismussion The IgE sensitized E-MC incubated for 15 min with heparan sulfate or heparin GAGs, showed an increase in antigen induced generation of LTC 4 in a dose dependent fashion (Fig 1). One million of IgE sensitized E-MC challenged with DNP-BSA released 20 ng ±2 (mean ± S.E., n = 5) of LTC 4 and 4.6 ng ± 0.3 (mean ± S.E., n = 5) of LTB 4. The maximum percent increase in LTC 4 generation in cells treated with 0.2 ~g heparan sulfate was 52 ± 3% (mean ± S.E., n = 5) (Fig. 1). Whereas 0.5 ~g of the GAG increased the production of LTB 4 by 50% (Table I). Ten micrograms of heparin slightly increased the LTC 4 production (33%) whereas lower doses were found to be ineffective (Fig. I).
I00
75 c
5O
u C o rc
"2
N
25
.,.1
"6
0.05
0.1
0.2
2
5
10
20
GLYCOSAMINOGLYCAN (Hg/ml. ) FIG.I Dose response effects of heparan sulfate •. . . . . . 0, heparin m ....... m and squid chondroitin sulfate-E A ....... A on the production of LTC 4. Each point represents the mean ± S.E. of 5 separate experiments, whereas according to t test p < 0.005.
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907
No significant increase in LTC 4 production was demonstrated when the IgE sensitized B-MC were treated with chondroitin sulfate-E GAG prior to the antigen challenge. Neither one of the three GAGs, at the various doses used, affected the antigen induced exocytosis of B -hexosaminidase from the IgE sensitized E-MC. Furthermore, a dose response of DNP-BSA varied from 0.8 ng to 500 ng/lO 6 cells was performed in the presence of lO ~g/ml of each of the GAGs. Neither one of the GAGs changed the pattern of the antigen dose dependent B-hexosaminidase release from the E-MC. In order to determine whether heparan sulfate GAG affects the production rather than the release of LTC 4, the cell associated residual LTC 4 was examined after antigen challenge. 6.8 ng ± 0.4 ng LTC4/IO 6 B-MC (mean ± S.E., n = 3) remained associated with the cell pellet of the heparan sulfate treated cells, whereas 7.1 ng ± 0.3 ng (mean ± S.E., n = 3) remained with the pellets of the untreated cells. Since the supernatant of heparan sulfate treated cells contained 66% increase in LTC4, and both the treated and untreated pellets remained the same, it may indicate that heparan sulfate pharmacologically regulates the synthesis of leukotrienes. The dose related effect of preincubation of IgE sensitized E-MC with heparan sulfate GAG on the antigen induced release of PGD 2 were determined in three separate experiments (Table I). After 15 min preincubation with heparan sulfate, antigen induced release of PGD 2 from 1 x lO 6 sensitized cells was inhibited from 5.4 ng ± 0.i into 3.5 ng • O.1 at 0.5 ~g/ml GAG.
TABl~ I Effect of heparan sulfate GAG on the PGD 2 and LTB 4 production by IgE sensitized E-MC stimulated with antigen Heparan sulfate ps/ml
0
0.i
0.5
1
PGD 2 ng/lO 6 cells
5.4 + O.1
4.1 + 0.08
3.5 + O.1
3.4 + 0.2
LTB 4 ng/lO 6 cells
4.6 + 0.3
5.7 ± 0.2
6.9 + O.1
6.7 + 0.3
* Means ± S.E. of five separate experiments.
Stimulation of B-MC with 0.05 uM and 0.2 uM calcium ionophore A23187 in the presence of each of the three GAGs at concentrations of 0.05 ~g to 20 ug, revealed that the amount of LTC 4 generation and the percent release of ~-hexosaminidase were in the same range of that obtained from cells treated by calcium ionophore alone (Table II).
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TABLE If Effect of GAGs on the LTC 4 production by E-MC stimulated with calcium ionophore A23187"
Heparan S u l f a t e
+ -
Hep~-In
+ -
~on~oitin Sulfat~E
+ -
A23187 ~ M
LTC4 n8/lO 6 cells
0.05 0.2 0.05 0.2
25.6 ± 1.0 70.3±2.3 24.8±0.8 69.7±7.6
13.0±0.8 25.6±0.4 15.0 ± 1.0 25.4±0.6
0.05 0.2 0.05 0.2
23.0 ± 2.0 72.7±3.8 22.7 ± 3.0 74.0 ± 1.7
16.0 ± 1.3 36.5±2.8 15.0 ± 1.9 38.6 ± 2.0
0.05 0.2 0.05 0.2
12.0 53.8 13.0 54.0
13.0 38.5 12.0 43.7
± ± ± ±
1.0 2.9 0.8 2.5
Net % R e l e a s e o f B- h e x ~ d a s e
± ± ± ±
1.1 2.7 2.0 1.5
*Mean ± S.E. of three separate experiments for each of lO ~g GAGs/ml. The % spontaneous B-hexosaminidase release from the unstimulated cells was 8.3% (mean ± S.E., n = 6)
It seems that heparan sulfate partially affected the shifting of arachionic acid from the cyclooxygenase to the 5-1ipoxygenase pathway which resulted in an increased production of leukotrienes and inhibition of PGD 2 synthesis. This slight modulation affected by heparan sulfate could not be observed when the cells were triggered with calcium ionophore A23187. This type of stimulator probably activates the 5-1ipoxygenase in E-MC by a different mechanism in comparison to the immunological stimulus. The homogenous heparan sulfate used in this study is a GAG with an apparent M.W. of 20,000 (6). The purification procedure for the heparan sulfate included extraction with 2 M NaC1 in order to separate electostatic bound proteins on the GAG. The GAG migrated as a single band on cellulose acetate strip electrophoresis. A very small concentration (0.2 ~g/ml) of heparan sulfate was found to be effective in enhancing the leukotiene synthesis (Fig. 1). Therefore, the possibility that contaminant molecules are those responsible for the enhancement effect seem to be unlikely. This finding is of particular interest, especially since both heparan sulfate and heparin GAGs do not have membrane effects that infulence the bridging response of specific IgE by antigen as determined by their uneffectiveness on the degranulation process.
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Heparin Sulfate and 5-Lipoxygenase
909
The evidence that heparin and its closely related GAG, heparan sulfate can be specifically bound to nucleated cells (i0) may indicate a unique mechanism by which mammalian cells modulate activation of leukotriene synthesis. Cultured mouse E-MC contain around 2.3 ~g granular chondroltln sulfate-E proteoglycan, whereas mouse peritoneal mast cells contain 3 u g granular heparin proteoglycan (11). Heparan sulfate is the predominant surface GAG in rapid dividing cells, especially in cancer lines (12). Several forms of heparan sulfate appear to be present, differing in size, charge density and ratio of N-acetyl to N-sulfate. More than one type of heparan sulfate was identified in embryonic mouse cells (13). It has been suggested that such changes may affect cellular metabolism by altering the calcium-binding capacity of the cell surface (14). Although the endogenous level of heparan sulfate in the mast cells was not determined yet the shedding of this component into the microenvironment from a variety of cell types and its binding to other cell suface may regulate the 5-1ipoxygenase activity by a mechanism which has to be further investigated. The chondroitin sulfate-E GAG is structurally different from heparin and heparan sulfate GAG. The disacccharides of which chondroitin sulfate GAG are composed differ from that of heparin, both in their uronic acid constituent (glucuronic acid rather than iduronic acid) and in their amino sugar, which is N-acetylgalactosamine-4-sulfate or 4,6-disulfate rather than N-sulfated glucosamine with or without an O-sulfate at the sixth carbon (15). Thus, the inability of chondroitin sulfate-E to modulate the LTC 4 synthesis is related to its GAG structure. References
I. 2. 3. 4. 5. 6. 7. 8. 9.
E. RAZIN, J-M. MENCIA-HUERTA, R.A. LEWIS, E.J. COREY and K.F. AUSTEN, Proc. Natl. Acad. Sci. U.S.A. 7 9 4665-4667 (1982). E. RAZIN, J-M. MENCIA-HUERTA, R.L. STEVENS, R.A. LEWIS, F-T. LIU, E.J. COREY and K.F. AUSTEN, J. Exp. Med. 157 189-201 (1983). E. RAZIN, L-C. ROMEO, S. KRILIS, F-T. LIU, R.A. LEWIS, E.J. COREY and K.F. AUSTEN, J. Immunol. 133 938-945 (1984). J-M. MENCIA-HUERTA, E. RAZIN, E.W. RINGEL, E.J. COREY, D. HOOVER, K.F. AUSTEN and R.A. LEWIS, J. Immunol. 3 0 1885-1890 (1983). C. RODEN, The Biochemistry of Glycoprotelns and Proteoglycans, W. Lennarz, (ed), pp. 267-371, Plenum, New York (1980). S. SCHILLER, G.A. SLOVER and A. DORFMAN, J. Biol. Chem. 236 983-987 (1961). Y. KAWAI, N. SENO and K. ANNO, J. Biochem. 6__00317-324 (1966). L. LEVINE, R.A. MORGAN, R.A. LEWIS, K.F. AUSTEN, D.A. CLARK, A. MARFAT and E.J. COREY, Proc. Natl. Acad. Sci. U.S.A. 787692-7696 (1981). R.A. LEWIS, J-M. MENCIA-HUERTA, R.J. SOBERMAN, et al., Proc. Natl. Acad. Sci. U.S.A. 797904-7908 (1982).
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I0. D.T. FEARON, Clinics In Immunology and AllerRy, A.S. Fauci (ed), pp. 225-242, W.B. Saunders Company Ltd., London, (1981). ll. E. RAZIN, R.L. STEVENS, F. AKIYAMA, K. SCHMID and K.F. AUSTEN, J. Biol. Chem. 257 7229-7236 (1982). 12. E.V. CHANDRASEKARAN and E.A. DAVIDSON, Cancer Res. 39870-880 (1979). 13. K.L. KELLER, C.B. UNDERHILL, and J.M. KELLER, Biochim. Biophys. Acta. 540 431-442 (1978). 14. S. VANNUCCHI, M. DEL-ROSSO, C. CELLA, P. URBANO and D.V. CHIARUGI, Biochem. J. 170 185-187 (1978). 15. H. SAITO, T. YAMAGATA and S. SUZUKI, J. Biol. Chem. 243 1536-1542
(1968).
Pergamon Journals
NEW ROLE FOR HEPARAN SULFATE: REGULATOR OF LEUKOTRIENE GENERATION IN MOUSE E-MAST CELLS M. Shalit,*
H. Shoam,**
N. Seno,*** and E. Razin**
*The Department Of Medicine, Hadassah University Hospital, **The Institute for Biochemistry, Hebrew University-Hadassah Medical School Jerusalem,lsrael ***Department of Chemistry, Ochanomizu University, Tokyo, Japan. (Received in final form June i0, 1986) Summary In this work, bovine heparan sulfate, pig mucosa heparin and squid chondroitin sulfate-E glycosaminoglycans (GAGs) were compared as to their affect on the synthesis of leukotrienes C 4 (LTC4) and B 4 (LTB4) as well as on the production of prostaglandin D 2 (PGD2) in cultured mouse E-mast cells (E-MC). The maximum percent increase in LTC 4 generation in cells treated with 0.2 ~g heparan sulfate was 52 ± 3% (mean ± S.E., n = 5). Whereas 0.5 ~g of the GAG increased the production of LTB 4 by 50% Ten micrograms of heparin slightly increased the LTC 4 production (33%) whereas lower doses were found to be ineffective. No significant increase in LTC 4 production was demonstrated when the IgE sensitized E-MC were treated with chondroitin sulfate-E GAG prior to the antigen challenge. Neither one of the three GAGs, at the various doses used, affected the antigen induced exocytosis of ~-hexosaminidase from the IgE sensitized B-MC. After 15 min preincubation with heparan sulfate, antigen induced release of PGD 2 from 1 x 106 sensitized cells was inhibited from 5.4 ng ± 0.I ng into 3.5 ng ± 0.i ng at 0.5 ~ g/ml GAG. All of the three types of GAGs used were uneffective when the E-MC were activated by calcium ionopore A23187. Upon stimulation by IgE-antigen or calcium ionophore A23187, cultured mouse E-~C release preformed mediators and produce oxidative metabolites of arachidonic acid predominantly through the 5-1ipoxygenase pathway (1,2,3). Leukotriene C 4 (LTC4) and B 4 (LTB4) are two of the major components of the 5-1ipoxygenase pathway within the mouse E-MC (1,4). The mechanisms for regulating leukotriene synthesis are not fully understood. This study focused on the role of heparan sulfate and heparin GAGs in the regulation of leukotriene synthesis in cultured mouse E-MC. Heparan sulfate is closely related to heparin GAG. It is characterized by a higher content of N-acetyl groups than is heparin while the content of N-sulfate groups are lower (5). Its predominant uronic acid is glucoronic acid, as opposed to iduronic acid in heparin. The degree of O-sulfation is lower and Correspondence: Dr. Ehud Razin, The Institute of Biochemistry, Hebrew University-Hadassah Medical School, P.O.Box 1172, Jerusalem, Israel. 0024-3205/86 $3.00 + .00 Copyright (c) 1986 Pergamon Journals Ltd.
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corresponds to that of the iduronic acid and N-sulfate content (5). From a biological viewpoint, heparin is characteristically stored in the granule of mast cells, from which it can be released in response to certain stimuli. Heparan sulfate proteoglycan, on the other hand, is an ubiquitous component of cell surfaces of many or all cell types (5). This study demonstrates that heparan and heparin GAG effectively regulate the leukotrienes synthesis after IgE-antigen stimulation of mouse E-MC. However, they were uneffective when the cells were activated by calcium ionopore A23187.
Materials and Methods Materials: Male BALB/C mice (Hebrew University animal breeding center, Jerusalem); WEHI-3 cell line (3); RPMI--1640, L-glutamine, non-essential amino acids, penicillin and streptomycin (Grand Island Biological Co., Grand Island, NY); fetal calf serum (FCS) (Biological Industries, Kibbutz Beit Haemek Israel); B-2-mercaptoethanol (Fisher Scientific, Medford, MA); P--nitrophenyl-B-D-acetamido--2-deoxyglucopyranoside; pig mucosa heparin (Sigma Chemical Co., St. Louis, MO); bovine heparan sulfate purified by chromatography (6) (Seikagaku Kogyo, Tokyo, Japan); squid chondroitin sulfate-E (8), calcium ionophore A23187 (Calbiochem-Behring Corp, La Jolla, Ca); 3H-LTC4 3H-LTB 4, and 3H-prostaglandin D 2 (PGD 2) (New England Nuclear Corp. Boston, MA) synthetic leukotrienes and antibodies against them (kindly provided by Dr. J. Rokach Merck, Canada); synthetic PGD 2 and antibodies against it (kindly provided by Dr. L. Levine, Boston, MA); mouse monoclonal anti-dinitrophenyl IgE (DNP) and DNP coupled to bovine serum albumin (DN-P--BSA) (kindly provided by Dr. F.-T. Liu, Medical Biology Institute, La Jolla, CA). Differentiation and sensitization of mouse E-MC: Bone marrow cells obtained from femurs of 2 month old BALB/C mice were cultured at 37°C in a humidified atmosphere containing 5% CO 2 at a starting density of 0.I x lO 6 cells/ml in RPMI 1640 supplemented with 10% FCS, 2 mM L-glutamine, O.1 mM nonessential amino acids, lO0 U/ml penicillin, lO0 ~g/ml Streptomycin, and 50 ~M B-2-mercaptoethanol at pH 7.2 (enriched medium); The enriched medium was supplemented with 50% WEHI--3 conditioned medium (WEHI-3-CM). WEHI-3-CM was produced by seeding WEHI--3 cells at 1 x 106/ml into the enriched medium and incubating them for 4 days at 37°C in 5% CO 2. Every 7 days the nonadherent cells from the bone marrow cultures were transferred into fresh enriched medium containing 50% WEHI--3-CM. The cultures were maintained for 14 days to obtain a F.-MC population of 97% purity~ as assessed by histological staining (3). Replicate samples of 1 x lOv E-MC were suspended in 0.2 ml of Tyrode's buffer containing 1 mM Ca 2+, 0.2 mM Mg 2+ and 0.05% gelatin (TG) and were sensitized by incubation for 1 hr at 37°C with 5 ~g/lO b cells of mouse monoclonal anti DNP IgE (2,3). Modulation of the activation response: Sensitized cells were washed with 1.5 ml of TG and sedimented at 400 x g at room temperature, they were then suspended in 400 ~l of TG
Vol. 39, No. i0, 1986
Heparin Sulfate and 5-Lipoxygenase
905
containing a defined concentrations of heparan sulfate or heparin or chondroitin snlfate-E GAGs and were incubated for 15 min at 37°C. 50 ng of DNP--BSA in I00 ~I TG were added to each sample and the incubation was continued at 37°C for I0 more minutes. Unsensitized cells (i x 106 ) in 400 ~i TG containing defined concentrations of each GAG were preincubated for 15 min at 37 ° C and activated for 30 min at 37°C by the addition of i00 ~i TG containing calcium ionophore A23187 final concentration of 0.05 and 0.2 ~M. All experiments were carried out in duplicate and included unchallenged controls for both sensitized and unsensitized cells. Cell viability was greater than 95% at the highest concentration used for each GAG. This was determined by trypan blue exclusion before as well as after antigen or ionophore activation. The antigen and ionophore activated cells were sedlmented at 400 x g for 5 mln at 22°C, and the supernatants were harvested. The cell sediments were resuspended in 500 BI of TG and sonicated at 4°C wi=h a Sonifier. Mediator assays: 8-Hexosaminidase was 2-deoxy-glucoparanoside : I 37°C (3). The results are B-hexosaminidase calculated
assayed by hydrolysis of p-nitrophenyl-B-DU of enzyme cleaves to I- mol of substrate/hr at expressed as the net percentage of release of according to the formula:
net percent release
S-Scontrol (S + P) - Scontrol
in which S = enzyme content of supernatant from stimulated cells. P = enzyme content of sediment from stimulated cells and Scontrol = enzyme content of supernatant from unstimulated cells. Radioimmunoassay (RIA) for the LTC 4 and PGD 2 was performed as described (8). RIA for LTB 4 was performed as described (9). In addition, duplicate samples of 400 ~I supernatants from the activated cells were mixed with 4 vol of methanol for 30 min at 4°C, centrifuged at 1500 x g for 5 min at 4°C to remove precipitated proteins. The extractions were either introduced on to Reverse Phase High Performance Liquid Chromatography (RP-HPLC) to detect leukotrienes, or dried and reconstituted with chloroform:methanol (2:1). Then the samples were loaded on thin layer plates (silica gel G 250 um, Brinkman), and developed in water-saturated ethyl acetate:2,2,4-trimethylpentane:acetic acid (110:50:0.8) together with PGD 2 standard. The PGD 2 fractions were scraped, extracted with methanol, dried and reconstituted with the RIA medium and the amount of PGD 2 was determined by the RIA. The leukotrienes were resolved by RP-HPLC with an isocratic solvent of methanol:water:acetic acid (65:34.9:0.1, pH 5.6) at a flow rate of 1 ml/min. Fractions were evaporated to dryness and redissolved in 500 ~i of the RIA medium for measurement of their leukotriene's immunoreactivity. The various GAGs did not interfere with the various RIA. This was determined after
906
Heparin Sulfate and 5-Lipoxygenase
Vol. 39, No. i0, 1986
comparison of the standard curve performed with or without the addition of the GAGs. In order to quantitate the LTC 4 remaining in the pellet of IgE-antigen stimulated cells incubated with or without lO ~g heparin or heparan sulfate, the pellets were mixed with 4 vol of methanol, incubated for 30 min at 4°C and centrifuged at 1500 x g for lO mins at 4°C to remove precipitated proteins. Each supernatant was evaporated to dryness, redissolved in 0.5 ml saline and assayed for LTC 4 by the RIA.
Results and Dismussion The IgE sensitized E-MC incubated for 15 min with heparan sulfate or heparin GAGs, showed an increase in antigen induced generation of LTC 4 in a dose dependent fashion (Fig 1). One million of IgE sensitized E-MC challenged with DNP-BSA released 20 ng ±2 (mean ± S.E., n = 5) of LTC 4 and 4.6 ng ± 0.3 (mean ± S.E., n = 5) of LTB 4. The maximum percent increase in LTC 4 generation in cells treated with 0.2 ~g heparan sulfate was 52 ± 3% (mean ± S.E., n = 5) (Fig. 1). Whereas 0.5 ~g of the GAG increased the production of LTB 4 by 50% (Table I). Ten micrograms of heparin slightly increased the LTC 4 production (33%) whereas lower doses were found to be ineffective (Fig. I).
I00
75 c
5O
u C o rc
"2
N
25
.,.1
"6
0.05
0.1
0.2
2
5
10
20
GLYCOSAMINOGLYCAN (Hg/ml. ) FIG.I Dose response effects of heparan sulfate •. . . . . . 0, heparin m ....... m and squid chondroitin sulfate-E A ....... A on the production of LTC 4. Each point represents the mean ± S.E. of 5 separate experiments, whereas according to t test p < 0.005.
Vol. 39, No. i0, 1986
Heparin Sulfate and 5-Lipoxygenase
907
No significant increase in LTC 4 production was demonstrated when the IgE sensitized B-MC were treated with chondroitin sulfate-E GAG prior to the antigen challenge. Neither one of the three GAGs, at the various doses used, affected the antigen induced exocytosis of B -hexosaminidase from the IgE sensitized E-MC. Furthermore, a dose response of DNP-BSA varied from 0.8 ng to 500 ng/lO 6 cells was performed in the presence of lO ~g/ml of each of the GAGs. Neither one of the GAGs changed the pattern of the antigen dose dependent B-hexosaminidase release from the E-MC. In order to determine whether heparan sulfate GAG affects the production rather than the release of LTC 4, the cell associated residual LTC 4 was examined after antigen challenge. 6.8 ng ± 0.4 ng LTC4/IO 6 B-MC (mean ± S.E., n = 3) remained associated with the cell pellet of the heparan sulfate treated cells, whereas 7.1 ng ± 0.3 ng (mean ± S.E., n = 3) remained with the pellets of the untreated cells. Since the supernatant of heparan sulfate treated cells contained 66% increase in LTC4, and both the treated and untreated pellets remained the same, it may indicate that heparan sulfate pharmacologically regulates the synthesis of leukotrienes. The dose related effect of preincubation of IgE sensitized E-MC with heparan sulfate GAG on the antigen induced release of PGD 2 were determined in three separate experiments (Table I). After 15 min preincubation with heparan sulfate, antigen induced release of PGD 2 from 1 x lO 6 sensitized cells was inhibited from 5.4 ng ± 0.i into 3.5 ng • O.1 at 0.5 ~g/ml GAG.
TABl~ I Effect of heparan sulfate GAG on the PGD 2 and LTB 4 production by IgE sensitized E-MC stimulated with antigen Heparan sulfate ps/ml
0
0.i
0.5
1
PGD 2 ng/lO 6 cells
5.4 + O.1
4.1 + 0.08
3.5 + O.1
3.4 + 0.2
LTB 4 ng/lO 6 cells
4.6 + 0.3
5.7 ± 0.2
6.9 + O.1
6.7 + 0.3
* Means ± S.E. of five separate experiments.
Stimulation of B-MC with 0.05 uM and 0.2 uM calcium ionophore A23187 in the presence of each of the three GAGs at concentrations of 0.05 ~g to 20 ug, revealed that the amount of LTC 4 generation and the percent release of ~-hexosaminidase were in the same range of that obtained from cells treated by calcium ionophore alone (Table II).
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Heparin Sulfate and 5-Lipoxygenase
Vol. 39, No. i0, 1986
TABLE If Effect of GAGs on the LTC 4 production by E-MC stimulated with calcium ionophore A23187"
Heparan S u l f a t e
+ -
Hep~-In
+ -
~on~oitin Sulfat~E
+ -
A23187 ~ M
LTC4 n8/lO 6 cells
0.05 0.2 0.05 0.2
25.6 ± 1.0 70.3±2.3 24.8±0.8 69.7±7.6
13.0±0.8 25.6±0.4 15.0 ± 1.0 25.4±0.6
0.05 0.2 0.05 0.2
23.0 ± 2.0 72.7±3.8 22.7 ± 3.0 74.0 ± 1.7
16.0 ± 1.3 36.5±2.8 15.0 ± 1.9 38.6 ± 2.0
0.05 0.2 0.05 0.2
12.0 53.8 13.0 54.0
13.0 38.5 12.0 43.7
± ± ± ±
1.0 2.9 0.8 2.5
Net % R e l e a s e o f B- h e x ~ d a s e
± ± ± ±
1.1 2.7 2.0 1.5
*Mean ± S.E. of three separate experiments for each of lO ~g GAGs/ml. The % spontaneous B-hexosaminidase release from the unstimulated cells was 8.3% (mean ± S.E., n = 6)
It seems that heparan sulfate partially affected the shifting of arachionic acid from the cyclooxygenase to the 5-1ipoxygenase pathway which resulted in an increased production of leukotrienes and inhibition of PGD 2 synthesis. This slight modulation affected by heparan sulfate could not be observed when the cells were triggered with calcium ionophore A23187. This type of stimulator probably activates the 5-1ipoxygenase in E-MC by a different mechanism in comparison to the immunological stimulus. The homogenous heparan sulfate used in this study is a GAG with an apparent M.W. of 20,000 (6). The purification procedure for the heparan sulfate included extraction with 2 M NaC1 in order to separate electostatic bound proteins on the GAG. The GAG migrated as a single band on cellulose acetate strip electrophoresis. A very small concentration (0.2 ~g/ml) of heparan sulfate was found to be effective in enhancing the leukotiene synthesis (Fig. 1). Therefore, the possibility that contaminant molecules are those responsible for the enhancement effect seem to be unlikely. This finding is of particular interest, especially since both heparan sulfate and heparin GAGs do not have membrane effects that infulence the bridging response of specific IgE by antigen as determined by their uneffectiveness on the degranulation process.
Vol. 39, NO. I0, 1986
Heparin Sulfate and 5-Lipoxygenase
909
The evidence that heparin and its closely related GAG, heparan sulfate can be specifically bound to nucleated cells (i0) may indicate a unique mechanism by which mammalian cells modulate activation of leukotriene synthesis. Cultured mouse E-MC contain around 2.3 ~g granular chondroltln sulfate-E proteoglycan, whereas mouse peritoneal mast cells contain 3 u g granular heparin proteoglycan (11). Heparan sulfate is the predominant surface GAG in rapid dividing cells, especially in cancer lines (12). Several forms of heparan sulfate appear to be present, differing in size, charge density and ratio of N-acetyl to N-sulfate. More than one type of heparan sulfate was identified in embryonic mouse cells (13). It has been suggested that such changes may affect cellular metabolism by altering the calcium-binding capacity of the cell surface (14). Although the endogenous level of heparan sulfate in the mast cells was not determined yet the shedding of this component into the microenvironment from a variety of cell types and its binding to other cell suface may regulate the 5-1ipoxygenase activity by a mechanism which has to be further investigated. The chondroitin sulfate-E GAG is structurally different from heparin and heparan sulfate GAG. The disacccharides of which chondroitin sulfate GAG are composed differ from that of heparin, both in their uronic acid constituent (glucuronic acid rather than iduronic acid) and in their amino sugar, which is N-acetylgalactosamine-4-sulfate or 4,6-disulfate rather than N-sulfated glucosamine with or without an O-sulfate at the sixth carbon (15). Thus, the inability of chondroitin sulfate-E to modulate the LTC 4 synthesis is related to its GAG structure. References
I. 2. 3. 4. 5. 6. 7. 8. 9.
E. RAZIN, J-M. MENCIA-HUERTA, R.A. LEWIS, E.J. COREY and K.F. AUSTEN, Proc. Natl. Acad. Sci. U.S.A. 7 9 4665-4667 (1982). E. RAZIN, J-M. MENCIA-HUERTA, R.L. STEVENS, R.A. LEWIS, F-T. LIU, E.J. COREY and K.F. AUSTEN, J. Exp. Med. 157 189-201 (1983). E. RAZIN, L-C. ROMEO, S. KRILIS, F-T. LIU, R.A. LEWIS, E.J. COREY and K.F. AUSTEN, J. Immunol. 133 938-945 (1984). J-M. MENCIA-HUERTA, E. RAZIN, E.W. RINGEL, E.J. COREY, D. HOOVER, K.F. AUSTEN and R.A. LEWIS, J. Immunol. 3 0 1885-1890 (1983). C. RODEN, The Biochemistry of Glycoprotelns and Proteoglycans, W. Lennarz, (ed), pp. 267-371, Plenum, New York (1980). S. SCHILLER, G.A. SLOVER and A. DORFMAN, J. Biol. Chem. 236 983-987 (1961). Y. KAWAI, N. SENO and K. ANNO, J. Biochem. 6__00317-324 (1966). L. LEVINE, R.A. MORGAN, R.A. LEWIS, K.F. AUSTEN, D.A. CLARK, A. MARFAT and E.J. COREY, Proc. Natl. Acad. Sci. U.S.A. 787692-7696 (1981). R.A. LEWIS, J-M. MENCIA-HUERTA, R.J. SOBERMAN, et al., Proc. Natl. Acad. Sci. U.S.A. 797904-7908 (1982).
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I0. D.T. FEARON, Clinics In Immunology and AllerRy, A.S. Fauci (ed), pp. 225-242, W.B. Saunders Company Ltd., London, (1981). ll. E. RAZIN, R.L. STEVENS, F. AKIYAMA, K. SCHMID and K.F. AUSTEN, J. Biol. Chem. 257 7229-7236 (1982). 12. E.V. CHANDRASEKARAN and E.A. DAVIDSON, Cancer Res. 39870-880 (1979). 13. K.L. KELLER, C.B. UNDERHILL, and J.M. KELLER, Biochim. Biophys. Acta. 540 431-442 (1978). 14. S. VANNUCCHI, M. DEL-ROSSO, C. CELLA, P. URBANO and D.V. CHIARUGI, Biochem. J. 170 185-187 (1978). 15. H. SAITO, T. YAMAGATA and S. SUZUKI, J. Biol. Chem. 243 1536-1542
(1968).