Cystic fibrosis serum promotes [45Ca] uptake by normal human leukocytes

Vol. 84, No. 4, 1978

BIOCHEMICAL

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

Pages

October 30, 1978

CYSTIC FIBROSIS SERUM PROMOTES [45Ca] BY NORMAL HUMAN LEUKOCYTES Martin W. Banschbach, Albert Department of Biochemistry

922-927

UPTAKE

G. Karam, Paula K. Love and Molecular Biology

and Bettina Department

C. Hilman of Pediatrics

The Louisiana State University Medical School of Medicine in Shreveport Shreveport, Louisiana 71130 Received

September

Center

11,1978

SUMMARY A two-fold increase in [ 45Cal labeling was observed when normal human leukocytes were incubated in the presence of serum obtained from patients with cystic fibrosis. The degree of t4'Cal labeling of leukocytes isolated from patients with cystic fibrosis was also significantly greater than that observed for leukocytes that had been isolated from normal individuals. INTRODUCTION A perturbation responsible

for

(CF) serum bioassay

on cilia

in

in

(3),

we decided

calcium

uptake

of

and cyclic high

beating

in

Since

transport

levels

using

the

normal

trachea of

intracellular

in

than

ionized

Press, Inc.

form reserved.

CF serum

in

the

922

since

(5).

is

of CF celluup-

et al.

re-

cyclic

AMP

leukocytes

of cells

calcium

on the [45Cal

less

do normal

and a de-

etiology

Davis

cilia

on calcium

been examined

produce

some types

fibrosis

explant

human leukocytes.

examined

may be

of cystic

of CF serum

CF leukocytes

B-agonists

AMP production

0 1978 by Academic of reproduction in any

effect

effect

0006-291X/78/0844-0922$01,00/0 Copyright All rights

rabbit

has not

the

was also that

with

of

the

of calcium

effect

may be involved

to evaluate [ 45Ca]

transport

dyskinetic

human cells

demonstrated

when stimulated

cellular

ciliary

by CF leukocytes

cently

the

(1,2).

normal

fect

take

the

system

transport

lar

in

inhibited

(4) by

BIOCHEMICAL

Vol. 84, No. 4, 1978

AND BIOPHYSICAL

RESEARCH COMMUNICATIONS

METHODS Leukocytes were isolated from 1 ml of whole heparinized blood, drawn by venipuncture from three CF patients and three Serum was obnormal subjects as described by Davis et aZ. (4). tained from 4 ml of whole blood, drawn by venipuncture from three CF patients and three normal subjects using a plastic syringe. All blood samples were drawn after obtaining informed signed consent. The three CF patients were recruited from the Shreveport Cystic Fibrosis Treatment Center. DP, an 11 year old male was taking sulfisoxazole (55 mg/kg/day) and sodium dicloxacillin BS, a 19 year old male was taking sodium oxa(72 mg/kg/day). cillin (179 mg/kg/day), cephalexin (36 mg/kg/day) and tetracycline (18 mg/kg/day). LG, a 14 year old male was taking sodium cloxacillin (117 mg/kg/day) and tetracycline (29 mg/kg/ Normal subjects were recruited from among the staff and day). faculty of the LSU Medical Center in Shreveport. MWB was a 31 year old male, PKL was a 25 year old female and LTG was a 38 None of the three normal subjects had taken any year old male. medication within 48 hours prior to the time that blood was drawn. Whole blood was allowed to clot at room temperature for 30 min. in a 15 ml plastic centrifuge tube and then centrifuged at 1,800 x g for 5 min. One ml of serum was removed from each sample and stored at room temperature for approximately 30 min. in a plastic test tube. After isolation, leukocytes were washed once in 4 ml of 0.9% NaCl (w/v) (centrifugation at 500 x g for 5 min.) and then resuspended in 4 ml of 0.9% NaCl (w/v) which contained 2.5 mMolar CaCl,. Each suspension of leukocytes was then split to yield two 2 ml fractions which were placed in 15 ml conical plastic centrifuge tubes. Then 0.5 ml of normal or CF serum was added to each tube followed by 30 ul of [45Ca]C1 (0.5 mCi) obtained from New England Nuclear, Boston, MA (17 mEi/ for 5 sec. and allowed to sit at mg) . Each tube was vortexed room temperature for 1 hour. Every 5 min., the contents of each The time period between the drawing of tube was gently mixed. blood for leukocyte isolation and the end of the incubation was approximately 2 hours. After the incubation, leukocytes were isolated by centrifugation at 1,000 x g for 5 min. and then washed one time in 4 ml of 0.9% NaCl (w/v) which contained 2.5 mMolar CaCl,. Then 5 ml of liquid scintillation cocktail developed by Anderson and McClure (6) for the solubilization of cultured cells was added to each tube and the cells were vortexed until completely dissolved. Beta emissions from [ 45Ca] were counted using a Packard Tri-Carb Model 3375 three channel liquid scintillation counter. Quenching was determined using the external standard channels ratio method but corrections for quenching were not made since all samples displayed essentially the same degree of quenching. RESULTS Data presented

in Table

icantly

stimulates

cytes.

Data presented

sence of CF serum,

1 demonstrate

the uptake

of

in Table

CF leukocytes

that

CF serum signif-

[45Ca]

by normal

2 indicate

that

take

923

human leuko-

even in the ab-

up significantly

more

Vol. 84, No. 4, 1978

BIOCHEMICAL

AND BIOPHYSICAL

Table Effect

of CF Serum on [ 45Ca]

Normal

Leukocytes and Normal Serum

1.

Uptake

By Normal

Human Leukocytes

Normal

Leukocytes and CF Serum

(cpm)

Difference

(cpn-4

34,345

(MWB-MWB)

36,889

(PKL-PKL)

23,950

(LTG-LTG)

31,728

RESEARCH COMMUNICATIONS

(MWB-DP)

74,669

75,500

(PKL-BS)

38,611

54,526

(LTG-LG)

30,576

109,014

f 3,958

(cpm)

79,680

+ 15,867"

47,952

f 13,558+

Leukocytes obtained from three normal human subjects were incubated in 2 ml of 0.9% NaCl containing 2.5 mMolar CaCl, with 0.5 mCi [ 45Ca] plus 0.5 ml of serum obtained from the same normal subject or 0.5 ml of serum obtained from a CF patient as described in the text. Initials in parenthesis designate individuals from whom leukocyte and serum samples were obtained. The bottom row of data represent the mean 5 S.E.M. for each set of data. *P < 0.05 using a one-tailed +P < 0.025 using a one-tailed

["Cal

than

do normal

leukocytes served

in for

serum

the

the

(compare

leukocytes.

presence

normal data

two sample unpaired two sample paired

The uptake

of CF serum

leukocytes in Tables

of

student's student's

[45Ca]

was similar

when they

were

to

t-test. t-test.

by CF that

exposed

ob-

to CF

1 and 2).

DISCUSSION Current

evidence

may alter

cellular

transport

of calcium

tivity

could that

metabolism

is tightly

a defect

in

the

cellular for observed

that via

(1,2). regulated

be responsible is routinely

suggests

a factor

present

a disturbance

Since

exocrine

in gland

by intracellular of calcium

producing

the

these

924

the

levels

ac(7),

in CF patients

exocrine

patients

cellular

secretory

calcium

transport

in

in CF serum

gland (8).

dysfunction

The data

pre-

BIOCHEMICAL

Vol. 84, No. 4, 1978

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

Table Uptake

of

2.

[ 45Ca] By CF Leukocytes

Normal Leukocytes and Normal Serum

CF Leukocytes and Normal Serum

34,345

(MWB-MWB)

85,678

(BP-MWB)

36,889

(PKL-PKL)

69,236

23,950

(LTG-LTG)

31,728

+ 3,958

CF Leukocytes and CF Serum

100,381

(DP-DP)

(BS-LTG)

76,437

(BS-BS)

65,644

(LG-PKL)

64,969

(LG-LG)

73,519

+ 6,167"

80,596

2 10,432+

Leukocytes obtained from three normal human subjects or three CF patients were incubated in 2 ml of 0.9% NaCl containing 2.5 molar CaCl, with 0.5 mCi [ 45Ca] plus 0.5 ml of serum obtained from normal subjects or CF patients as described in the text. Initials in parenthesis designate individuals from whom leukocytes and serum samples were obtained. The bottom row of data represent the mean + S.E.M. for each set of data. *P < 0.01 using a one-tailed +P < 0.025 using a one-tailed test.

sented

in this

paper

serum can promote The data

also

even

in the

that

observed

agreement strate

tive

demonstrate

the uptake

of

that

tb5Ca]

for

normal

data

CF fibroblasts

this

greater

of a greater cyclic

intracellular

then

expected

to be depressed

t-test. t-

in CF

human cells.

by CF leukocytes, greater

observation

et

up significantly

al.

which

than is

in

demon-

more [4sCa]

(9).

uptake

calcium,

by Shapiro

take

fibroblasts

by normal

This

student's student's

something

is significantly

leukocytes.

presented

that

[ 45Ca] uptake

absence of CF serum,

than do normal If

clearly

demonstrate

with

that

two sample unpaired two sample unpaired

of

[ 45Ca] by CF leukocytes pool

AMP production

of both by adenyl

in CF leukocytes

925

if

is

indica-

bound and unbound cyclase ionized

might

be

intracellu-

Vol. 84, No. 4, 1978

lar

calcium

does

in

some other

ulated

Most

effect also

according

the

penecillin-type

the

sulfonamides the

not

skin

indicate

biopsies

fibroblasts turing

information

mine Wilson

bioassay

studies

experiments on this

in CF patients. if

manufacturers of

but

low and during

effects

on the

and

demonstrated not

re-

of CF leukocytes not

indicate samples

Shapiro

et aZ.

antibiotics

antibiotic

be very

of

(11)

when blood

taking

and

action

are

(1,2).

were

(9),

antibiotic

an ionophoreic

et al 0 did

cilia

patients

currently

when

levels

in

the

cell

cultured

cells

skin culwould

be insignificant.

Additional

calcium

Bogart

were taking

probably

via

AMP response

patients

their

not

et aZ.

their

were obtained

process,

probably

(4).

if

are

and cloxacillin cyclic

Some

tetracyclines

Davis

tetracycline

would

the

rep-

antibiotics

on the mechanism

In addition,

the

the

by the

(lo),

which

patients.

study

supplied

reviews

(12).

these

this

antibiotics

system

activities

antibiotics

for

was when stim-

on oral

However,

antimicrobial

stimulation

were obtained

recently

leukocytes

pulmonary

among

for

to literature

antibiotics

et al.

as it

in CF leukocytes

placed

death

selected

depressing

B-agonist

Davis

of the

of

to recent

for

in leukocytes

to normal

as ionophores.

antibiotics

sponsible

which

cause

their

according

(5).

routinely

infection

patients

known to exert

activity

AMP production

are

may act

by the

RESEARCH COMMUNICATIONS

(4).

bacterial the major

taken

cyclase

compared

CF patients

antibiotics

did

cyclic

B-agonists

to prevent

AND BIOPHYSICAL

of cells

depressed

with

resents

types

that

significantly

that

adenyl

inhibits

demonstrated

to

BIOCHEMICAL

the

ciliary

and Fundenberg

are

apparent

being

defect

in

An attempt dyskinesia (13)

conducted

will

factor is the

926

agent

the

to provide

cellular

transport

be made to try isolated

from

responsible

more

to deter-

CF serum for

of

by

producing

BIOCHEMICAL

Vol. 84, No. 4, 1978

this If

[ 45Ca] uptake a defect

occur

effect

responsible

for

transport

then

inducing

RESEARCH COMMUNICATIONS

of CF serum on normal

in the cellular

in CF patients,

AND BIOPHYSICAL

this

of

calcium

exocrine

human leukocytes.

calcium

does indeed

transport

gland

defect

dysfunction

may be

in these

patients. ACKNOWLEDGEMENTS This R. Stiles

work was supported Trust Fund.

by a grant

obtained

from

the Edward

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Bogart, B.I., Conod, Res. 11, 131-134.

2.

Bogart, (1978)

3.

Ochsman, J.L. (1976) Calcium Ions Conference Report, Cystic Fibrosis

4.

Davis, P.B., 12, 703-707.

5.

Rasmussen, H., Jensen, Goodman, D.B.P. (1975) 375-394.

6.

Anderson, 173-179.

7.

Schreurs, Bonting,

8.

Changus, J.E. 100, 7-11.

and Pitot,

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Shapiro, Warwick,

Feigal, R.J., (1978) Clinica

B.I., Pediat.

E.J.

and Conover,

Conod, E.J., Gaerlan, Res. 12, 15-24.

Braunstein,

L.E.

J.H.

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and Conover,

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Wilkowske,

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Neu, H.C.

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