Prostaglandins: Action on mast cells in vitro

PROSTAGLANDINS:

P.

ACTION ON MAST CELLS IN VITRO.

Albro, R. Thomas and L. Fishbein

National Institute of Environmental Health Sciences National Institutes of Health, Public Health Service and Department of Health, Education, and Welfare Research Triangle Park, North Carolina 27709

Prostaglandins Al, Bl, El, E2 and Fla at concentrations between 10-7 and lo-SF1failed to induce release of histamine from rat peritoneal ma3 cells. PGEl and PGE2 stabilized mast cells against spontaneous histamine release, but had only a slight inhibitory effect on histamine release induced by 48180. Calcium chloride and oxidized fatty acid salts did induce histamine release to some extent, but dibutyryl cyclic-AMP did not.

Accepted Feb. 23, 1972.

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PROSTAGLANDINS

Introduction Crunkhorn and Willis (1) have reported the use of Pontamine blue 6BX dye as an indicator of increased vascular permeability after cutaneous injection of prostaglandins. They theorized from the results of experiments in which mast cell histamine was depleted by 48/80 or antagonized with mepyramine and methysergide prior to injection of prostaglandins, that prostaglandins might be "trigger substances" in the release of mast cell histamine. Pickles(2), in a review article on prostaglandins, quoted Cabut and Vincenzi (3) as reporting that PGEl at a concentration of 500 mug/ ml caused disruption of rat mast cells in vitro with release of histamine and heparin. However, when we atErnmto locate the original work of Cabut and Vincenzi, we found that it had in fact not been published and therefore no experimental details were available. Arora et al. (4) stated that PGEl disrupts mast cells, citing a reference wF&in fact made no mention of any effect of prostaglandins on mast cells (5). Juhlin and Michaglsson (6) and Michaglsson (7) state that "the role of histamine is not essential for the formation of an erythematous response to PGEl". Mannaioni (8) reported that PGEl did not affect either release or uptake of histamine by murine neoplastic mast cells in vitro. Kaley and Weiner (9) stated that PGEl did not cause disruptionmast cells in rat mesentery pieces in vitro. These conflicting reports suggested to us that it would be useful to reevaluate the relationship between prostaglandins and release of histamine from mast cells. The association of prostaglandins with the anaphylactic response (10) remains unexplained, although Koopman et al. (11) have shown that both PGEl and PGE2 inhibit the IgE- and GGa-mediated release of slow-reacting-substance of anaphylaxis in the rat. Further information on the relationship between prostaglandins and histamine release will hopefully clarify this subject. Materials and Methods Histamine hydrochloride and dibutyryl 3',5'-cyclic adenosine monophosphate (DB-cyclic-AMP) were from Sigma. Prostaglandins were kindly provided by Dr. John Pike, Upjohn Company. Compound 48/80 was donated by the Burroughs, Wellcome Company. Cobra (Na'a nigricollis) venom was from Pierce Chemical Co. Fatty acids and --+ ot er lipids were from Applied Sciences Laboratories. Sprague-Dawley (CD) rats weighing 250-3509 were anae'sthetized with Metofane and decapitated. Each received an intraperbitoneal injection of 6 ml of physiological saline pH 7.0 containing 0.1% bovine serum albumin (fraction V, Armour), after which total per'itoneal

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were recovered as described by Johnson and Moran (12). Mast cells were purified by centrifugation through a 35% solution of bovine serum albumin for 10 minutes at 250 x g (13). Purified mast cells were washed with and final1y suspended in saline - 0.1% albumin pH 7.0; the suspensions contained approximately 0.5-l pg of total histamine per ml. Cell susoensions from several rats were pooled prior to setting up the incubations described below.

cells

In a few cases mast cells were isolated from peritoneal fluid without the 0.1% albumin and incubated in saline alone. One experiment was performed using total peritoneal cells in order to check for possible cooperative interactions with mixed cell types. Incubations were carried out in 15 ml centrifuge tubes. Each tube received 2.0 ml of cell suspension and 10 microliters of a saline or 50% aqueous ethanol solution of the substance being tested. In control experiments, up to 0.1 ml of 50% ethanol could be added to cell suspensions without causing histamine release or interfering with the release induced by 48/80 or cobra venom. Samples were treated with the test substances, mixed well and incubated at 37O for 5 minutes. They were then cooled to 4O with an ice bath, centrifuged at 250 x g to sediment the mast cells, and assayed for histamine release. Histamine was assayed by a modification of the o-phthaldehyde method of Shore et al. (14). The same fluorometric results were obtained whether anintact cell pellet or the supernate after complete disruption with distilled water at 62O (15) were processed with or without the butanol extraction step. A single peak of fluorescence having excitation and emission maxima at 350 and 450 nm, respectively, was always observed using an Aminco-Keirs Spectrophosphorimeter in the fluorescence mode. This observation made it possible to omit the butanol extraction step (which only recovers 85% of the histamine)(14), but it was still necessary to correct for the 20% suppression of fluorescence caused by the sodium chloride in the incubation mixtures (16). These were the onlv modifications we made in the assav I Drocedure I of Shore -_* et al (14). " The centrifuged incubation mixtures were separated into 1.0 ml of supernatant and 1.0 ml of supernatant plus cells. Each portion was assayed for histamine. The amount released was taken as twice the amount in the 1 ml of supernatant, while the amount left in the cells was taken as total minus amount released. None of the compounds used as potential histamine releasers interfered with the histamine assay at the concentrations involved here. Results When mast cells were kept in the presence of 0.1% albumin, spontaneous histamine release was less than 3% through the duration

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of the experiments. When albumin was not used in collecting peritoneal cells or during the incubations, spontaneous histamine relgase reached 60%. This henomenon has been previously discussed by Uvnas and Thon (17,18P. The effects of prostaglandins on spontaneous histamine release in the absence of albumin is shown in Table I. E orostaalandins aooarentlv had dose-dependent stabilizing effects under these conditions. 'PGEl and PGE2 were equally potent at the 10-6~ level, but PGFlcrand PGAl showed no significant effect. Unfractiona ed peritoneal washings did not release histamine when treated with lo-sy PGEl, although they released 90% of their histamine when treated with 48/80. PGEl but not DB-cyclic AMP slightly inhibited the 48/80-induced histamine release. The addition of glucose did not lead to release of histamine in the presence of PGEl (Table II). Glucose was included in some of these tests as it is reportedly helpful in maintaining normal metabolic activity in the mast cells (18). Most of the tests were performed using purified mast cells in saline with 0.1% albumin, as summarized in Table III. For the purposes of this table the amounts of histamine released were corrected for spontaneous release and compared to the amounts released by 48/80 set equal to 100% (actually 7580% of the total histamine in the cells). The first portion of Table III shows that the stabilizing effect of prostaglandins against spontaneous histamine release seen earlier was not very evident when measured against 48/80-induced histamine release from purified mast cells. The role of the 0.1% albumin in this observation is uncertain. -7 PGAl, PGBl, PGEl, PGE2 or PGFla at concentrations between 10 and lo-6M did not cause significant histamine release under the conditions of-these experiments. These observations apply to freshly prepared solutions of prostaglandins; we have occasionally and unreproducibly found histamine releasing activity (up to 20%) in solutions of PGE2 allowed to stand for several days at 40. This may have involved some oxidation, dehydration or hydrolysis product of the prostaglandin, but we have not been able to observe the effect consistently and have not examined it in detail. Autooxidized linolenic acid and the hydroxy acids ricinoleic and deoxycholic show a similar effect (Table III) but at much higher concentrations. The last section of Table III shows that CaC12 may have released histamine from mast cells and that the effect, if real, was concentration dependent. However, the release was very slight up to lo-2! calcium and not increased by the inclusion of prostaglandins. DBcyclic-AMP did not relea e histamine in these experiments. Inclusion of divalent cations (Mg+3 , Cat2). dibutyryl cyclic AMP, or glucose in the incubation media along with prostaglandins did not result in increased histamine release, nor did addition of PGEl already in salt form (K).

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TABLE I.

Effect of Prostaglandins on Spontaneous Histamine Release.a

Treatment

Final Concentration

Trials (Rats)

Average Percenta e Released8

S.D.+

52.7

5.6

Saline

0.15M

4 (8)

48180

25 vg/ml

4 (8)

PGE,

10_6!

3 (6)

36.3

4.2*

lo-6!j

3 (6)

51.4

3.8

PGA,

10-61

3 (6)

48.6

7.5

PGE2

10_6!

3 (6)

35.1

2.0*

PGE2

10_5!

3 (6)

4.9

1.0+

PGE2

5x10-51

3 (6)

6.1

2.1+

PGFIQ.

100

5.0

aFrom mast cells incubated without albumin (17). bNot corrected for spontaneous release; corrected for amount released by 48/BO set equal to 100%. *P < 0.05, 2-tailed t-test. t

P < 0.01, 2-tailed t-test.

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25 ug/ml 1 ug/ml (A) 10m5E, (B) 1 vg/ml (A) 10m31$ (B) 1 Pg/ml lo-51J (A) 10-3& lo-3y

48180

48180

(A) PGEl, (B) 48/80

(A) DB-cyclic-AMP, (B) 48/80

PGEl

(A) glucose, (B) PGEl

DB-cyclic-AMP

2

2

2

2

2

2

4

4

Trials

0

0

0

75.7

68.9

79.9

100

0

When two compounds

0.8

0.1

0.1

1.2

1.7

0.3

3.3

-

Range, !:

bCorrected for spontaneous release (= 5%) and for the amount released by 25 ug 48/8Cl per ml set equal to 100%.

aIncubated in saline-0.1% albumin. Substances added in 10 ~1 of solvent. were added, (A) was added 1 min. before (B).

(B) 10-5y

10 ~1/2 ml

Final Concentrations

Average Percentage Releasedb

Histamine Release from Total Peritoneal Cell~.~

Saline or 50% ethanol

Treatment

TABLE II.

3 2 UJ

E

2

8 g

25 ,g/ml 10-51 10_5! 10_7! 10_6! lo-5fi 5x10-6! lo-6PJ lo-5fi lo-6y

Cobra venom

PGAl

PGBl

PGEl

PGEl

PGEl

PGEl, potassium salt

PGE2

PGE2

PGFllI

(A) 10m6M_, (B) 25 ug/ml

(A) PGF,, (B) 48/80

(B) 25 Ug/ml

(A) 10-f&

25 pg/ml

48/%0

(A) PGE2, (B) 48/m

10 V1/2 ml

Final Concentration(s)

5

4

4

4

8

4

4

4

4

4

3

3

20

20

Trials

0

0

0.1

0

0.3

0

0

0

0

86.4'

94.6

99.4

100

0

Average Percentage Releasedb

Histamine Release from Albumin-Stabilized, Purified Mast Cells.

Saline or 50% ethanol

Addition(s) to Mediuma

TABLE III.

0.4

0.2

0.9

0.3

0.6

0.8

0.9

0.1

0.3

6.7

1.3

0.9

4.6

0.4

S.E.M.

’

2 CA

ZJ

5:

&

2

(Cont'd.)

(B) PGE2

(B) PGFla

(B) PGFla

(A) CaC12,

(A) CaC12,

(A) CaC12,

(A) 10-3bJ, (B) 10-5E

(A) 5~10-~!, (B) lO-%J

(A) 5~1O-~bJ, (B) lo+-bj

(A) 5~1O-~bJ, (B) lo-%J

4

4

4

4

(B) PGEl

(A) 5~1O-~bJ, (B) lO%J

(A) CaC12,

4

4

5x10-5!

DB-cyclic-AMP

4

(B) PGE2

3 pg/ml

Sodium Deoxycholate

3

(A) MgC12,

250 pg/ml

Sodium Ricinoleate

8

4

3 ug/ml

Sodium Ricinoleate

5

4

4

Trials

(A) DB-cyclic-AMP, (B) PGEl (A) 10m3fJ, (B) 10w5kj

250 vg/ml

(A) 10-31'J (B) 10-5E

lo-5M -

Final Concentration(s)

0.4

0

1.2

0.3

0

0

0

0.9

6.7

0.6

13.1

0..8

0.3

Average Percentage Releasedb

Histamine Release from Albumin-Stabilized, Purified Mast Cells.

Sodium Linolenated

(A) Glucose, (B) PGE,

PGFla

Addition(s) to Mediuma

TABLE III.

0.3

0.3

1.0

0.6

0.2

0.2

0.3

0.3

2.2

0.4

4.4

0.2

0.8

S.E.M.

3 Z cn

c

R

g 2

cd

(Cont'd.)

lo-3y 10-21

CaC12

CaC12 4

4

4

Trials

4.4

1.0

0.3

Average PercEntage Released

(A) added

1.3

0.2

0.1

S.E.M.

d

At pH 5.4, cobra venom released 2.8 times as much histamine as did 48/80.

Subjected to autooxidation.

'At pH 7.0.

Corrected for spontaneous release (~3%) and for release induced by 25 ug 4B/BO per ml set equal to 100%.

b

aCompounds added in 10 ~1 of saline or 50% ethanol to 2 ml of incubation mixture. 2 min. before (B).

!XX~O-~M

Final Concentration(s)

Histamine Release from Albumin-Stabilized, Purified Mast Cells.

CaC12

Addition(s) to Mediuma

TABLE III.

PROSTAGLANDINS

Discussion Mast cells purified through albumin and incubated in a very simple medium of 0.9% NaCl-0.1% BSA pH 7 constitute a suitable system for screening histamine releasers. This system retained 97+% of its cellbound histamine during the incubation of controls, was responsive to cobra venom, and released 75-80% of its total histamine under the influence of 25 vg of 48/80 per ml. These parameters compare closely to those of the more elaborate systems used by Uvn$s and Thon (17,18) and Johnson and Moran (12), suggesting that release of histamine from rat peritoneal mast cells in vitro does not require exogenous glucose, phosphate, potassium orcalcium. It is possible, however, that endogenous stores of some or all of these are retained during the purification used here and are fully adequate for histamine release. Contrary to the report quoted by Pickles (2), we find no evidence that prostaglandins act on mast cells to release histamine in vitro. We find, rather, that PGE's stabilize mast cells against spontaneous histamine release. This was the case both in the presence and absence of albumin, glucose, calcium ion, and dibutyryl cyclic-AMP. There is one conspicuous difference between the actions of histamine releasers in vivo and -in vitro that may be relevant here. It has been pointed zt-(i-@ that release of histamine from mast cells by 48/80 does not require calcium ions in vitro, but does in vivo. This being the case, it may be that mast ~31s would be more cnmve to calcium ion concentration -in vivo than they were in the experiments reported here. Coceani and Wolfe (19) have suggested that prostaglandins may induce contraction of stomach smooth muscle through release of calcium from a stored complex. Prostaglandin has also been reported to release "loosely bound" calcium from the isolated rat uterus (20). It is thus tempting to speculate that the in vivo release of histamine thought by Crunkhorn and Willis (1) to resultfrom administration of PGEl might, if in fact it does occur, result from a local effect of prostaglandins on calcium ion concentration. Prostaglandin El and E2 but not Flc(have been reported to stimulate synthesis (or at least increase levels) of leukocyte cyclic AMP and also to inhibit antigenically induced release of histamine from these cells (21). In this case the inhibition by prostaglandin was thought to be a result of increased cyclic AMP levels, and again, was considered to operate during the Ca/Mg-independent phase of histamine release. In our experiments, we were unable to detect any effect of dibutyryl cyclic AMP. It is impossible, however, to rule out the possibility that DB-cyclic-AMP acts differently from -in vivo-synthesized cyclic AMP. Although our Table III is largely filled with negative results, we felt that a complete presentation was necessary in view of our original objective to determine, with some degree of confidence, whether or not prostaglandins have a direct action on mast cells

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resulting in release of histamine. From the data presented in Table III, we are convinced they do not. This initial study has not examined the relationship of prostaglandins, if any, to antigenically induced release of histamine from sensitized mast cells. Should subsequent study disclose an inhibitory effect of prostaglandins on this process analogpus to the stabilizing effect reported in the present paper, a role for prostaglandins in anaphylaxis would be clarified. References 1.

2.

3.

4. 5. 6.

7. 8. 9. 10. 11.

12. 13. 14.

Crunkhorn, P. and A. L. Willis. Cutaneous Reactions to Intradermal Prostaglandins. Brit. J. Pharmacol. 41:49, 1971. Pickles,V. R. The Prostaglandins. Biol. Revs. 42:614, 1967. Cabut, M. S. and L. Vincenzi, in Nobel Symp. 2. Prostaglandins (S. Bergst& and B. Samuelsson, Editors) Almquist and Wiksell, Stockholm, 1967. Arora, S., P. K. Lahiri, and R. K. Sanyal. The Role of Prostaglandin El in Inflammatory Process in the Rat. Int. Arch. Allergy 39:186, 1970. Horton, E. W. Action of Prostaglandin El on Tissues Which Respond to Bradykinin. Nature (London) 200:892, 1963. Juhlin, L. and G. Michaklsson. Cutaneous Vascular Reactions to Prostaglandins in Healthy Subjects and in Patients with Urticaria and Atopic Dermatitis. Acta Dermatovener (Stockholm) 49:33, 1969. Michaglsson, G. Effects of Antihistamines, Acetylsalicylic Acid and Prednisone on Cutaneous Reactions to Kallikrein and Prostaglandin El. Acta Dermatovener (Stockholm) 50:31, 1970. Mannaioni, P. F. Influence of Bradykinin and Prostaglandin El on the Uptake and Release of Histamine by Murine Neoplastic Mast Cells in vitro. Biochem. Pharmacol. 19:1159, 1970. Kaley, G. and R. Weiner. Prostaglandin El: A Potential Mediator of the Inflammatory Response. Anal. N. Y. Acad. Sci. 180:338, 1971. Piper, P. J. and J. R. Vane. Release of Additional Factors in Anaphylaxis and Its Antagonism by Anti-inflammatory Drugs. Nature 223:29, 1969. Koopman, W. G., R. P. Orange, and K. F. Austen. Prostaglandin Inhibition of the Immunologic Release of Slow Reacting Substance of Anaphylaxis in the Rat. Proc. Sot. Exptl. Biol. Med. 137:64, 1971. Johnson, A. R. and N. C. Moran. Comparison of Several Methods for Isolation of Rat Peritoneal Mast Cells. Proc. Sot. Exptl. Biol. Med. 123:886, 1966. Lagunoff, D. and E. P. Benditt. 5-Hydroxytryptophan Decarboxylase Activity in Rat Mast Cells. Am. J. Physiol. 196:993, 1959. A Method for Shore, P. A., A. Burkhalter, and V. H. Cohn, Jr. the Fluorometric Assay of Histamine in Tissues. J. Pharmacol. Exptl. Therap. 127:182, 1959.

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15. 16. 17. 18. 19. 20. 21.

144

Bray, R. E. and P. P. VanArsdel, Jr. In vitro Histamine Release from Rat Mast Cells by Chemical and Phzimgents. Proc. Sot. Exptl. Biol. Med. 106:255, 1961. Michaelson, A. and H. R. Smithson. Effects of Halogens on oPhthaldehyde-Histamine and Spermadine Fluorescence. Anal. Chem. 43:1300, 1971. Uvn&, B. and I. Thon. Isolation of "Biologically Intact" Mast Cells. Exptl. Cell Res. 18:512, 1959. Uvnis, B. and I. Thon. Evidence for Enzymatic Histamine Release from Isolated Rat Mast Cells. Exptl. Cell Res. 23:45, 1961. Coceani, F. and L. S. Wolfe. On the Action of Prostaglandin El and Prostaglandins from Brain on the Isolated Rat Stomach. Can. J. Physiol. Pharmacol. 44:933, 1966. Paton, 0. M. and E. E. Daniel. On the Contractile Response of the Isolated Rat Uterus to Prostaglandin El. Can. J. Physiol. Pharmacol. 45:795, 1967. Bourne, H. R., L. M. Lichtenstein, and K. L. Melmon. Leukocyte Cyclic Adenosine Monophosphate Inhibits Antigenic Histamine Release. J. Clin. Invest. 50:10a, 1971.

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Albro, P., R. Thomas, and L. Fishbein Prostaglandins: Action on Mast Cells In Vitro REVIEWERS' COf+tENTS The most important result, the failure of PGEl to release histamine from peritoneal mast cells (rat) in vitro, has already been published (Loeffler, et al, Biochem. Pharm. 20:2287, 1971). The authors add a number of other prostaglandins to this list, useful, but by no means startling information. The relative insensitivity of their albumin-protected cells to release by 48/80 (25 ug/ml vs. 1 vg/ml in the study cited above) suggests that the simplified saline solution (pH 7.0, but what buffer was used?) used by Albro, et al, may not be a useful system for screening histamine releasers (contraryto the first sentence of the discussion). If their cells truly are relatively insensitive in this buffer system, the conclusion that prostaglandins fail to release histamine is weakened. The failure of dibutyryl cyclic AMP to inhibit histamine release is at variance with the results of Loeffler, et al, who showed inhibition of 48/80 release at a slightly lower concentration of the cyclic AMP analog. Since Loeffler, et al, also showed an inhibitory effect on histamine release by PGEl, it seems likely that Albro, et al, are using an insensitive or unphysiological mast cell preparation which requires too much 48/80 for release. The phosphate, divalent ions, etc. are probably more important than the authors think. The question asked by the authors is an interesting one, and a solidly negative answer would be important (i.e. prostaglandins do not cause release of histamine from mast cells in vitro). Unfortunately, this conclusion has in part (with PGEl) been documented before, and problems with methodology vitiate the potential validity of the new data with other prostaglandins.

AUTHORS' REPLY Apparently in opposition to the reviewers' opinion, we saw (and see) no reason to assume that any conclusions concerning the ability of prostaglandins to release histamine from mast cells could be drawn from results obtained using only a single prostaglandin. We were aware that Loeffler, et al, had published the results of some experiments similar to ours, but unfortunately our library did not receive a copy of that issue of Biochem. Pharm.

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in time for a reference to it to be included in our manuscript. We would agree with the reviewer that some reference to Loeffler, et al, should be made. There was nothing in our paper indicating that a 48/80 concentration of 25 pg/ml was required for histamine release. In fact, maximum histamine release was reached at approximately 2.5 pg of 48/80 per ml. Our Table II shows that 1 pg of 48/80 per ml released approximately 65% of the total histamine; in the preparations used by Loeffler, et al, 1 pg of 48/80 per ml released 52% of the total histamine in 2 experiments, 75% in another. One of our attempts to find inhibition by PGEl of the 48/80-induced histamine release was, as Table II shows, performed using 1 ug of 48/80 per ml. Loeffler, et al, probably saw more inhibition of 48/80induced histamine release than we did simply because they preincubated with PGEJ for 30 minutes compared to our one minute. We felt that the persistence of prostaglandins in an animal after their release is probably quite short, and therefore we did not want to emphasize effects requiring long-term exposure to prostaglandins in the present study. As discussed above, we feel that the evidence in our paper indicates that our preparations were at least as sensitive to 48/80 as were those used by Loeffler, et al. We routinely used 25 pg of 48/80 per ml to insure that our data could be expressed in terms of "percentage of maximum releasable histamine." In regard to dibutyryl cyclic AMP, there may be two reasons why Loeffler, et al, saw inhibition and we did not. First, they were using total peritoneal cells, while we were using purified mast cells. It is already known (our reference 21) that cyclic AMP inhibits histamine release from leukocytes. Second, once again they preincubated with cyclic AMP for a much longer time than we did.

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