Comparison of circulating and peritoneal leukocyte cationic proteins in release of histamine from rat mast cells

Life Sciences Vol. 13, pp. 1117-1130, 1973. Printed in Great Britain

Pergamon P r e s s

COMPARISON OF CIRCULATING AND PERITONEAL LEUKOCYTE CATIONIC PROTEINS IN RELEASE OF HISTAMINE FROM RAT MAST CELLS Ronald G. Coffey Sloan-Kettering Institute for Cancer Research New York, New York 10021

(Received 17 May 1973; in final f o r m 4 September 1973) SUMMARY Lysosomal cationic proteins which release histamine from rat peritoneal mast cells were prepared from circulating as well as peritoneal leukocytes of rabbits. The release of histamine by cationic proteins and by compound 48/80 was compared as a function of temperature, pH and concentration. Cationic protein-medlated histamine release appears to be a non-cytotoxlc energy requiring process similar to compound 48/80-medfated release. It was inhibited by iodoacetate, n-ethylmaleimlde, 2,4-dlnltrophenol, malonate, oxamate, glutamate and slightly inhibited by 2-deoxyglucose. Pharmacologic inhibition of release by isoproterenol, amlnophylllne, dibutyryl cyclic AMP and prednisone was also demonstrated. Introduction Cationic proteins capable of causing inflammatory changes have been extracted from rabbit and guinea-pig peritoneal polymorphonuclear granules by several workers.

leukocyte

Zeya and Spitznagel (I) described the anti-

bacterial properties of some of these proteins from guinea-pig leukocytes. Jonoff and Seegers (2,3) purified a specific rabbit leukocyte cationic protein (LCPm)* of low molecular weight and high arginine content that causes degranulatlon and histamine release from rat mast cells.

Keller et al. (4,5) con-

firmed their findings, and found that this purified LCPm did not possess bactericidal properties.

Ranadlve and Cochrane (6,7) demonstrated that the

purified LCPm causes rat mast cell degranulation by an energy requiring mechanism similar to that of both compound 48/80 and homocytotropic antibody. *The abbreviation LCPm is used here to denote exclusively that leukocyte cationic protein which causes release of histamine from mast cells, while LCP denotes leukocyte cationic proteins in general.

1117

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Leukocyte Cationic P r o t e i n s and Histamine

Vol. 13, No. 8

In this paper we show that LCPm can be prepared in good yields from circulating as well as peritoneal exudate rabbit leukocytes.

The prepara-

tions appear identical in the mechanism by which they provoke the release of histamine from rat mast cells.

The effects of several metabolic inhibitors

on LCPm-mediated histamine release are compared to compound 48/80-mediated

release. Materials and Methods Chemicals.

Glycogen (from shell fish, type II), ficoll (M W 400,000),

iodoacetate, n-ethylmalelmide, 2,4-dlnltrophenol, malonate, oxamate, glutamate, 2-deoxyglucose, prednlsone and isoproterenol were obtained from Sigma Chemical Co., St. Louis, Me.

Dextran (M W 200,000-300,000) was obtained from

Nutritional Biochemical Corp., Cleveland, Ohio, Sephadex G-25 (fine) from Pharmacia, Piscataway, N. J., aminopbylline from Mann Research Laboratories, N. Y., N. Y. and compound 48/80 from Burroughs Wellcome Co., Tuckahoe, N. Y. Preparation of Cells and LCPm.

Leukocyte and mast cell preparations

were performed with polyethylene or polypropylene ware.

Male Angora rabbit

peritoneal leukocytes were prepared by the procedure of Cohn and Hirsch (8) following a twelve hour instead of a four hour induction with 0.i g glycogen dissolved in I00 ml saline.

These preparations ranged from 90 to 95 percent

neutrophils. Peripheral leukocytes were then collected by cardiac puncture and separated from red cells by dextran sedimentation method of Lichtensteln and Osier (9).

Leukocytes were washed twlce with saline and centrifuged at

150 × g for i0 minutes.

These preparations ranged from 70 to 85 percent

neutrophils and less than one red cell or platelet per ten leukocytes. Leukocytes were homogenized at 0°C in 0.34 M sucrose using a motor driven glass pestle.

The homogenate was centrifuged at 0°C for I0 minutes at

5000 × g and the sediment consisting of nuclei, mitochondria and unbroken cells was discarded.

Lysosomal and other granules were then sedlmented at 0°C for

30 minutes at 15,000 × g.

This differential sedimentation technique does not

Vol. 13, No. 8

Leukocyte Cationic Proteins and Histamine

1119

separate LCP-containlng granules from those rich in lysosomal enzyme activities. LCPm was prepared by extracting the granules three times with cold 0.2 N H2S04, precipitated from the combined extracts with cold ethanol at a flnal concentration of 20 % (v/v), dlssolved in 0.05 M sodium acetate and 0.15 M NaC1, pH 4.1 and purified by gel filtratlon through a 0.9 × 60 cm column of Sephadex G-25 as described by Seegers and Janoff (3).

The degree of

purification was assessed by determinations of protein according to Lowry et al (10) and rat mast cell histamine releasing capacity as described below. Rat mast cells were obtained from the peritoneal cavities of Spraguet!

Dawley or Wistar rats by saline lavage according to Uvnas and Thon (Ii).

Mast

cells were stained with 0.05 Z toluldlne blue in saline at pH 7.0 and counted in a hemocytometer,

then centrifuged at room temperature for 5 minutes at

100 X g and resuspended in buffer so that one ml contained 105 mast cells. The buffer consisted of 1 3 8 m M Na CI, 2.5 mM KCI, 1.0 mM MgCI2, 0.9 mM CaCI2, 5 mM glucose and 13 mM K2HPO 4 - KH2PO4, adjusted to pH 7.25 with HCI. Histamine Release.

One tenth ml of mast cell suspension (104 cells) was

added to a 12 x 75 mm polypropylene tube containing 0.85 ml buffer and other experimental compounds.

Cells were incubated at 37°C under air with metabolic

or pharmaco]oglc Inhibltors for 5 minutes, then 0.05 ml of LCPm (approximately one unit as defined below) was added, and incubation was continued for another ten minutes.

One unit of LCPm was defined as that amount which provokes the

release of 50 percent of the histamine in 104 mast cells in one ml at 37°C during a ten minute incubation.

The tubes were then chilled In an ice bath

and centrifuged at 150 X g for 5 minutes at 0°C.

Histamine was determined in

both the supernatant and the cells by the fluorometric method of Shore et al (12) as modified by Lichtensteln and Osler (9).

A Turner Model i0 fluorometer

equipped with a high sensitivity attachment, a primary emission filter No. 760, and secondary filters No. 2A and 48 were used, providing mldrange readings for fifty ng histamine.

Catecholamlnes, methylxanthlnes and 2,4-dlnltrophenol

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Leukocyte Cationic P r o t e i n s and Histamine

Vol. 13, No. 8

which interfered with the assay were removed by butanol extraction

as des-

cribed by Shore et al (12). Percent histamine a =ng

histamine

control

release was calculated

in supernatant,

(no LCPm)

tube.

h =ng

Percent

as [a/(a+b)

in cells,

inhibition

percent

release with LCPm plus inhibitor.

percent

in all experiments.

Replicates

each condition or inhibitor. within 5 percent

indicated when 2 or more experiments

for the

release was calculated

release with LCPm and I ffi

Control release varied

from 5 to 8

of 3 or 4 tubes were performed

The deviations

and are not reported.

and c = a/(a+b)

of histamine

as i00 × (A - I)/A, where A ffi percent histamine

- c] X i00, where

among replicates

Standard deviations

with different

for

were usually

of the mean are

rats were averaged.

Results Purification

of LCPm.

LCPm from both peritoneal summarized

in Table I.

The preparation and circulating

The preparations

and 5.2 X 109 circulating

cells.

Yields

a per cell or per mg protein basis. was obtained

of active mast cell degranulatlng

leukocytes represent

is

7.6 X 109 peritoneal

cells

from either source are comparable

An apparent

for the LCPm from peritoneal

from 19 rabbits

purification

leukocytes

on

of 116 fold

and 50 fold from circula-

ting cells.

TABLE Preparation

I

of LCPm from Rabbit Leukocytes

Peritoneal

Cells

protein mg

Circulating units mK_

LCPm units

Cells

Fraction

LCPm units

Homogenate

2132

369

6

1730

289

6

Granules

1232

55

22

1220

79

15

0.2 N H2SO 4 extract

1162

28

28

40

11.6

50

42

1130

20% ethanol ppt.

520

8.5

61

582

Sephadex C~25 peak

250

0.36

695

260

protein mg

units m8

0.87 300

Vol. 13, No. 8

L e u k o c y t e C a t i o n i c P r o t e i n s and H i s t a m i n e

The Sephadex activity occurred cell sources.

filtration data are shown in Figure i. at 130 - 140 ml eluant

The final preparations

The peak LCPm

for both peritoneal

enzymic action on mast cells.

releasing

Following

activity,

incubation

with one unit LCPm from either source, mast cells appeared treated with 0.5 ug/ml compound

48/80.

ly visible under 400 x magnification, ing granules had been extruded. surface of the cells. selective, 48/80 (13). lyric factor"

factor"

of mast cells

identical

to those

cell membrane was clear-

Some of these granules

adhered

caused by LCPm appears

to the outer to be a

process similar to that caused by antigen or compound to refer to LCPm as a "mast cell

(MCLF, ref. 5) or "mastocytolytic

factor" and mast cell-rupturing

2,3).

/

0]0=

A 280

ruling out non-

while many of the toluidine blue stain-

It is probably not appropriate

(MCF, r e f .

040 -

The continuous

The degranulation

non-cytotoxic

and circulating

of LCPm from both sources were boiled

for five minutes with no loss of histamine specific

1121

.30

J 1

O.L>O=

• 20

Unit p ~ ml

z ,,

010 •

J \ ,o

&

I~D

160

190

~o

ml I l u l ~ l LCP {1~

&

~o ~

FIG.

~o

~o

L ~ (C)

1

Sephadex C-25 P u r i f i c a t i o n

of LCPm

LCPm, precipitated by 20% ethanol, was dissolved in 0.05 M acetic acid, 0.15 M NaCI (pH 4.1) and applied to a 0.9 x 60 on column of Sephadex G-25. Elution with the same buffer was performed at 4°C at 0.5 ml per minute. . . . . . , LCPm activity in units per ml; , protein estimated by absorbance at 280 n=.

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Leukocyte Cationic P r o t e i n s and H i s t a m i n e

Metabolic Requirements.

Vol. 13, No. 8

The dependence of LCPm-mediated histamine re-

lease on temperature and pH are shown in Table II and Figure 2, respectively. In these and subsequent experiments the Sephadex G-25 'peak' fractions for peritoneal

(P) and circulating

(C) leukocyte LCPm were used.

The optimum

conditions for release are similar to those described for compound 48/80 and for antigen (13,14) and appear identical here with those for compound 4 8 / 8 0

TABLE II Temperature Dependence of Histamine Release

Percent Histamine Release* Temperature

LCPm(P)

LCPm(C)

48/80

5 °C

5--+i

2±2

2_+I

37 °C

64 -+ 2

53 3_ ~ 2

76 -+ 2

45 °C

30 -+ i

28 .~. 1

41 -+ 0

*Data represent the means±deviations

from the mean for two experiments.

Cells were incubated at 5 °C or 37 °C with approximately one unit of LCPm or 0.5 ~g per ml of compound 48/80 for ten minutes, or preincubated at 45 °C for five minutes followed by ten minutes at 37 °C with releasor.

Histamine was

then determined in the supernatant and cells as described in Methods.

Dose responses to LCPm and compound 48/80 are shown in Figure 3.

The

curves indicate that both releasing agents provoke release of histamine as a function of the logarithm of concentration of releasing agent. Inhibition of LCPm-mediated histamine release by a variety of metabolic inhlbitors is shown in Table III.

The degree of inhibition by these compounds

was similar for either source of LCPm and for compound 48/80. was the most potent of the agents used.

N-ethylmaleimide

lodoacetate is often preferentially

used to block glycolysis at the 3-phosphoglyceraldehyde

dehydrogenase step,

while both compounds are effective inhibitors of active -SH groups.

Addition

Vol. 13, No. 8

Leukocyte Cationic P r o t e i n s and H i s t a m i n e

1123

80 =

60 = Pe-cenl of Moxlmum Release

40 =

20 =

pH

FIG. 2 pH Dependence of Histamine Release The degree of release was calculated as percent of the maximum observed at pH 7.25 for convenience in comparing the curves for peritoneal LCPm, O 0, circulating LCPm, O O , and compound 48/80, A A. Each point represents the mean of 3 experiments. Standard deviations (not shown) ranged from 0 to 3 percent histamine released.

of i0 m M c y s t e i n e

prevented inhlhition by both compounds.

oxldatlvephosphorylation, 2,4-dinitrophenol, at 2 mM.

this inhibition was reversed by i0 mM

Oxamate, a competitive inhibitor of pyruvate utilizing enzymes, was

less effective; glucose.

caused almost complete inhibition

Halonate, a competitive inhibitor of succinate dehydrogenase, was

an effective inhibitor at 2-5 ~ : glucose.

An uncoupler of

inhibition by this compound was also reversed by i0 TaM

Glutamate, which inhibits compound 48/80-mediated histamine release

(15) also inhibited LCPm-medlated release. requiring a concentration of I0 to 50 mM. equimolar concentration of glucose.

2-deoxyglucose was a poor inhibitor, This inhibition was reversed by an

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Leukocyte Cationic Proteins and Histamine

-T

~

Vol. 13, No. 8

j

l_oq~ o Un~

FIG. 3 Dose Response of Histamine

Release

to LCPm and Compound 48/80

Each point represents the mean of 2 experiments. Points were plotted with reference to the concentration of peritoneal LCPm, O-- . . . . ~, circulating LCPm, ~ O , or compound 48/80, 5. . . . . . . ~, which provided 50% release (i unit) of histamine at pH 7.25.

Inhibition of histamine

release by four pharmacologic

reported

in Table III.

Prednlsone was the most effective,

mediated

release by 40 percent at a concentration

of I0 ~M.

agents are also inhibiting Dibutyryl

cyclic AMP was the least effective of the four, while isoproterenol phylllne,

compounds

many tissues, concentrations.

capable of raising Intracellular

inhibited

release by about

LCPm-

and amino-

levels of cyclic AMP in

50 percent at 0.i to 0.5 ~M

Vol. 13, No. 8

Leukocyte Cationic P r o t e i n s and Histamine

1125

TABLE III Inhibition

of Histamine

Percent Concentration

Inhibitor

Release

Inhibition of Release Mediated by: LCPm(C) 48/80

LCPm(P)

mM

lodoacetate

*

0.2 1.0

26 ± I0 78 ± 6

29 ± 92 ±

0.05 0.20

67 ± 95 ±

5 2

57 ± 1 84 ± 14

2,4-dinitrophenol

0.5 2.0

39 ± 96 ±

7 5

34 ± 89 ±

5 6

Malonate

1.0 2.0 5.0

i0 ± 2 30 ± 7 70 ± I0

14 ± 31 ± 67 ±

4 9 7

]0.0 20.0

7 36

10 42

i0.0 20.0

35 60

30 57

5.0 i0.0 50.0

Ii 17 48

13 18 55

N-etbylmaleimlde

Oxama t e

*

**

**

Glutamate

2-deoxyglucose

**

3 3

15 ± 6 75 ± 13 70 ± 91 ±

5 3

28 54

Prednlsone

0.01

42 ±

6

39 ±

8

22 ±

Isoproterenol

0.i

52 ±

3

51 ±

5

40 ± 17

Amlnophylline

0.5

55

Dlbutyryl

1.0

26 ±

cyclic AMP

Histamine

cells

(LCPm(C)

in Methods.

separate experiments cysteine.

6

33 ±

21 5

release by one unit of LCPm from peritoneal

from circulating as described

45

7

41 ±

cells

7

(LCPm(P)

or 0.5 ~g compound 48/80 per ml was measured

Standard deviations

where indicated.

*

of the mean are reported

and by 5 0 m M

for two

Inhibition was reversed by I0 mM

** Inhibition was reversed by i0 mM glucose for 5 and i0 mM

2-deoxyglucose

or

glucose for 50 mM 2-deoxyglucose.

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Leukocyte Cationic Proteins and Histamine

Vol. 13, No. 8

Discussion Significance of Circulatin 8 LCP.

Zeya and Spitznagel

(i) reported in

1966 the presence of very small amounts of LCPm in circulating leukocytes of guinea-pigs following a four hour glycogen induction of peritoneal cells. Frimmer et al (16) later stated that basic lysosomal peptides had been found only in exudate leukocytes.

In 1970 Nachman et al (17) stated that no lysos-

omal cationic permeability activity had been detected in circulating leukocytes Zeya and Spitznagel

(1) suggested that circulating leukocytes might be capable

of elaborating the LCP in response to some triggering agent.

Janoff (2)

showed that a mast cell degranulating cationic protein, not easily demonstrable in circulating rabbit leukocytes, was "activated" and released into the medium following incubation with antigen-antibody bacterial endotoxin.

complexes or by exposure to

Subsequently Hegner (18) and Frimmer and Lutz (19) pre-

pared LCP in good yields from circulating bovine leukocyte lysosomes;

the

latter workers showed the capacity of the preparation to release histamine from rat mast cells and to increase the permeability

in rabbit skin.

In contrast to the work cited above, we prepared rat mast cell histamine releasing LCPm in good yields from circulating rabbit leukocytes.

The major

difference in our technique involved our use of a 12 hour rather than a 4 hour period of glycogen induction, after which the peripheral leukocytes were obtained by cardiac puncture.

It is possible that this longer period allowed

"activation" by endotoxin or other impurities in the glycogen, as suggested by Seegers and Janoff (2,3).

Zeya and Spitznagel

of LCP in immature rabbit bone marrow leukocytes,

(20) found a high content less LCP in exudate cells,

and very low content in mature peripheral leukocytes.

They suggested that

some form of secretion of specific granule product may occur during maturation and peripheral passage of the cells. The occurrence of histamlne-releasing LCPm in human leukocytes is questionable.

Janoff (2) stated that it could not be found.

Welsh and

Vol. 13, No. 8

Spitznagel

Leukocyte Cationic Proteins and Histamine

1127

(21) extracted LCP with bactericidal properties from circulating

human leukocytes, but the presence of the histamine-releasing LCPm was not demonstrated.

However, they suggested that methods used for isolation of

rabbit cationic proteins have restricted applicability in other species. Nachman et al (17) and Kelly et al (22) demonstrated a hlstamine-releaslng cationic protein in human platelets, but were unable to demonstrate its presence in leukocytes.

Kelly et al (22) observed the release of histamine from

human leukocytes by lysates of purified human lymphocytes and granulocytes, but they believed the active factor was not a cationic protein because it could not be precipitated with 20 percent ethanol.

On the other hand, Keller

(4) reported the presence of LCPm in leukemic leukocytes.

Dunn and $picer

(23) found that early neutrophils from human bone marrow contain small mucosulfate-rich granules similar to those of rabbit peritoneal leukocytes, while human neutrophils of later developmental stages have few or no such granules. Thus the significance of LCPm in humans may be restricted to conditions in which leukocytes prematurely enter the circulation. Mechanism of Action of LCPm.

Ranadive and Cochrane

(7) showed that the

rabbit leukocyte LCMm-mediated release of histamine from rat mast cells was an energy requiring process similar to histamine release induced by compound 48/80 or antigen. cells at 45°C

They demonstrated inhibition of release by heating mast

prior to addition of LCPm, incubating cells with LCPm at 5 °C,

and inhibition by 2,4-dinitrophenol

in the absence of glucose, by iodacetate

(glycolytic inhibitor) and by diisopropy] fluorophosphate inhibitor).

(serine esterase

We have confirmed their findings with regard to heat, cold in-

cubation, 2,4-dlnltrophenol and iodoacetate using both peripheral and circu]ating rabbit leukocyte sources of LCPm.

Inhibition by n-ethylmaleimide,

an

agent which blocks active -SH groups, was similar for both sources of LCPm and for compound 48/80.

In addition we observed some inhibition by malonate

competitive inhibitor of succinate dehydrogenase) and glutamate

(a

(unknown site

1128

L~ukoc~yte Cationic P r o t e i n s and H i s t a m i n e

of action).

Vol. 13, No. 8

The effects of 2-deo~yglucose were very slight, in agreement with

~akravarty's results using compound 48/80 or antigen (24). ~Jere seems to be little doubt that LCP provokes histamine release by a mechanism which involves activation of a serine esterase (6,7) and requires a source of cellular energy which can be derived from oxidative phosphorylation via the tricarboxylic acid cycle or from substrate level phosphorylation via the glycolytic reactions.

Histamine release is reduced in proportion to the

reduction of Intracellular ATP levels (25). Pharmacologic inhibition of antigen-lnduced histamine release by betaadrenergic stimulation of adenylate cyclase with isoproterenol has been demonstrated in human leukocytes (26) and human lung (27).

Baxter (28) re-

ported similar inhibition of dextran-induced histamine release from mast cells. In these studies theophylline, a cyclic AMP phosphodiesterase inhibitor, potentiated the effects of isoproterenol.

In the present study we report

inhibition by isoproterenol and amlnophylline (which forms 2 molecules of theophylllne in solution) and by dlbutyryl cyclic AMP and prednisone.

In

another study (29) we found that prednlsone as well as isoproterenol can increase cyclic AMP in flcoll-purified rat mast cells.

Whether this action

is related to the inhibition of histamine release remains to be determined. Pharmacologic regulation of histamine release may be mediated by effects on enzymes other than adenylate cyclase.

Kaliner et al (30) have demonstrated

augmentation of antlgen-medlated release of histamine from lung tissue by alphaadrenergic and cholinergic stimulation.

The former involves reduction of cyclic

AMP levels (30) and increased ATPase activity (29) while the latter causes increased cyclic GMP levels (31).

Current studies on the opposing effects of

the two cyclle nucleotldes on phosphorylation of mast cell enzymes may increase our understanding of the mechanism of histamine release and its pharmacologic regulation.

Vol. 13, No. 8

Leukocyte Cationic Prot ei ns and Histamine

Acknowledgement This work was supported in part by U.S. Public Health Service Grant No. AI-09619. References I.

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