Circulating

factors contribute

to elevation of intracellular monophosphate and depression of

cyclic-.3’,5’-adenosine superoxide

anion

following

production

thermal

Ann

Abstract:

B. Bjornson7

We

Scott

D. Somers

James

of Surgery,

have

N

University

previously

Robert

Gamble

Institute

of Cincinnati

demonstrated

tericidal activity and superoxide are depressed concomitantly in kocytes (PMNs) following thermal

that

College

bac-

anion (02) production polymorphonuclear leuinjury in a guinea pig

model, and the bactericidal defect is related to elevation of intracellular cyclic-3’,5’-adenosine monophosphate (cAMP). The purpose of the present investigation was to determine the relationship between elevation of intracellular CAMP and depression of #{176}2 production in PMNs following thermal injury and determine the involvement of circulating factors in the development of these alterations. The kinetics of 02 production and dose responses to formylmethionyl-leucyl-phenylalanine (fMLP) and phorbol myristate acetate (PMA) were depressed in peripheral PMNs following thermal injury in this cxperimental model. Sera obtained during the period of PMN dysfunction induced depression of 02 production in response to fMLP and elevation of intracellular CAMP in normal PMNs. Pretreatment of normal PMNs with nonsteroidal anti-inflammatory drugs (NSAID; indomethacin or piroxicam) inhibited the elevation of intracdllular CAMP mediated by sera from the injured animals but had no effect on the depression of #{176}2 production observed under similar conditions. Treatment of PMNs from injured animals with NSAID under conditions correct

known the

production. jured patients

to reduce bactericidal

the

CAMP defect

Studies utilizing sera confirmed findings

of serum-mediated depression of 02

elevation production

content did not

of the cells normalize

from two thermally in the guinea pig

of intracellular in normal PMNs

and #{176}2

inmodel

CAMP and and effects

observed with NSAID. These results suggest that circulating factors contribute to the elevation of intracellular CAMP and depression of #{176}2 production in PMNs following thermal injury. Whereas the increase in intracellular CAMP may be involved in the depression of 02 production, our results suggest that there is not a direct link between these alterations. J. Leukoc. Biol. 52: 407-414; 1992. Key Words: monophosp/zate

.

polymorphonuclear superoxide anion

leukocytes

injury

6Div#{252}ion of Immunology, Department

in polymorphonuclear

leukocyte production

.

.

cyclic-3’,5’ thermal

W. Knippenberg7 of Medical

Research

of Medicine,

and and

H. Stephen

Division

Cincinnati,

of Surgical

as

a consequence

of trauma.

suggesting that oxidative PMNs following thermal phagocytosis has generally creased The functional

[2, 12, 21, mechanisms responses

There

been

various

reports

22]. underlying of PMNs

the selective depression following thermal injury

of are

not well understood, and this information is important for the development of therapeutic approaches directed toward normalizing effector functions critical for host defense against infection. PMNs appear to be activated as a consequence of severe trauma as evidenced by increased expression of complement receptors (CR1 and CR3) [9], loss of specific granules [11], and increase in plasma lactofernin [23]. Defective chemotaxis of the PMNs in response to C5a has been attributed to down-regulation of C5a receptors [7, 10]. We have demonstrated that intracellular cyclic-3’,S’-adenosine monophosphate (cAMP) is markedly increased in PMNs folbowing thermal injury in a guinea pig model, and the elevation of intracellular cAMP plays a central robe in defective bactericidal activity of these cells against Pseudomonas aeruginosa [14]. Increased spontaneous production of prostaglandin E, (PGE,) by the PMNs was found to be an important mechanism beading to the elevation of intracellular cAMP. The bactericidal defect of PMNs was accompanied by depression of superoxide anion (02) production in response to fonmylmethionyl-leucyl-phenylalanine (IMLP). The present investigation was undertaken to determine the involvement of the elevation of intracellular cAMP in the depression ofO2 production in this model. Since circulating factors have been previously implicated in alterations of ox-

Abbreviations: tion; E,;

NSAID, PMA,

SOD, local and systemic production of and concomitant depression of vanthat are essential for host defense

have

metabolism also is depressed in injury [15-20]. In contrast, been reported to be normal or in-

CAMP,

cyclic

3’,5’-adenosine

formylmethionyl-leucyl-phenylalanine;

Thermal injury induces inflammatory mediators ous cellular responses

Diseases,

Ohio

against infection (reviewed in ref. 1). Polymorphonuclear leukocyte (PMN) dysfunction appears to play an important role in the increased susceptibility to bacterial infection associated with thermal injury [2-4]. The two effector functions of PMNs that have been studied extensively following thermal injury are chemotaxis [5-11] and bactericidal activity [2-4, 12-14]. Both functions have been shown to be depressed in thermally injured patients and animal models

-adenosine injury

INTRODUCTION

Bjornson Inftctiou.s

nonsteroidal phorbol

superoxide

acetate;

of

Leukocyte

drug;

PMN,

balanced

PGE,,

salt

solu-

prostaglandin

polymorphonuclear

leukocyte;

dismutase.

Reprint requests: Ann B. Bjornson, Gamble Institute of Medical Research, OH 45241. Received January 24, 1992; accepted

Journal

Hanks’

anti-inflammatory

myristate

IMLP,

monophosphate;

HBSS,

Biology

Division of Immunology, 2141 Auburn Avenue,

Volume

May

James Cincinnati,

N.

4, 1992.

52,

October

1992

407

Preparation

idative metabolism associated with thermal injury [17-20], the contribution of these factors to the development of PMN alterations also was investigated. We hypothesized that circulating factors produced in response to thermal injury might contribute to the elevation of intracellular cAMP in PMNs and this in turn might be responsible for the depression of O2 production. The results of our study support the concept that circulating factors contribute to the elevation of intracellular CAMP and depression of 02 production in PMNs following thermal injury; however, they suggest that there

is not

a direct

link

between

these

PMNs were prepared from the peripheral blood of guinea pigs [14] or humans [24] as previously described. The cells were suspended in Hanks’ balanced salt solution (HBSS) for measurement of02 production or HBSS containing 20 mM HEPES, pH 7.4, for determination of cAMP content. As reported previously, guinea pig preparations contained 85% PMNs and 15% mononuclear cells [14]. Human PMN preparations were 95% pure.

alterations.

Viability MATERIALS

AND

of PMNs

METHODS

of PMNs

Viability

was

Animals

animals

were

fed

guinea

pig

chow

ad

production

were

thermal

injury

injured

of

three injection was drawn

Determination PMNs

animals were anesthetized by of 30 mg/kg sodium pentobarbital, by cardiac puncture. The blood

in-

Journal

of Leukocyte

Biology

the

PMNs

broincubated of viability. of O2 with

serum.

were thawed immedion wet ice for up to

52,

October

(5.0

1

Measurement The

(1.8 x 10 cells/ml) were serum (v/v) or HBSS for cytochalasin B treatment. B, 1.5 mg/ml cytochrome concentrations of fMLP

of CAMP content x 106 cells/mI)

were

incubated

at 37#{176}Cwith

10%

periods. The cell and cAMP conas previously

1992

primary

of PGE metabolites PGE

metabolites

in

serum

(13,14-dihydro-15-

keto-PGE2, 13,14-dihydro-15-keto-PGA2, and PGA2 covalently bound to albumin) were converted to 11-deoxy-13,14dihydro-15-keto-llfl ,16e-cycbo-PGE2 (bicyclic PGE2), and this bicyclic compound was measured by radioimmunoassay using a kit from Amersham (Arlington Heights, IL). The manufacturer’s directions were followed for the conversion of PGE metabolites to bicyclic PGE2 and for the radioimmunoassay procedure. The antiserum to bicyclic PGE2 in the kit was 100% cross-reactive with bicyclic PGE,, and thus the results reflect the combined content of bicyclic PGE, and bicyclic

Volume

of O2 production

was

collected in glass tubes containing 5 sM mecbofenamate to prevent generation of prostaglandins in vitro. In some cxperiments, animals were injected intramuscularly with 10 mg/kg indomethacin, 15 mg/kg piroxicam, or placebo at 3 h postburn [13], and blood was collected at 1 day postburn. The blood was allowed to clot at room temperature and then was stored at 4#{176}Cfor up to 2 h. The tubes were centrifuged at l000g for 10 mm at 4#{176}C,and the sera were removed. Sera from injured and sham-treated animals were separately pooled and stored in small aliquots at - 70#{176}C.Human sera were prepared identically, except mecbofenamate was not in-

408

incubating

serum (v/v) or HBSS for the indicated time suspensions were then boiled and sonicated, tent was measured by radioimmunoassay described [14].

of sera

cluded during blood collection. Sera ateby before use or were maintained h before use.

for

after

or phorbol myristate acetate (PMA), and HBSS with or without 50 sg/ml superoxide dismutase (SOD) were added in a total volume of 200 sb. The reactants were incubated at 37#{176}Cfor the indicated time intervals, and the reaction was stopped by addition of 100 sb of HBSS containing 100 jzg/mb SOD to tubes lacking SOD and 100 sl of HBSS alone to tubes containing SOD. The tubes were centrifuged at l000g for 5 mm at 4#{176}C,and the absorbance ofthe supernatants was measured spectrophotometrically at 550 nm. SOD-inhibitable O2 production was quantitated using an extinction coefficient of 21.1 mM’ cnf’ for cytochrome c.

patients

and handling

serum before assessment below under measurement

on 02 production, the PMNs preincubated at 37#{176}Cwith 10% specified time intervals before the After treatment with cytochalasin C, 15 tg/ml catalase, the indicated

Sera were obtained within 18 to 24 h postburn from two patients with flame burn injuries hospitalized at the Burn Speciab Care Unit, University Hospital, University of Cincinnati Medical Center. Patient A was a 53-year-old female with a 35% total body surface burn (29% partial thickness, 6% full thickness). Patient B was a 52-year-old male with a 66% total body surface burn (48.5% partial thickness, 17.5% full thickness). Neither patient had clinical signs of sepsis at the time of serum collection. Informed consent was obtained from the patients or their guardians, and human experimentation guidelines of the U.S. Department of Health and Human Services and those of the authors’ institutions were fobbowed in the conduct of the clinical research.

Collection

used

microscopy

diacetate-ethidium the PMNs were

O2 production by PMNs was measured by a modification of the method of Cox et al. [26]. PMNs (1.0 x 106 cells) in HBSS were incubated with 1 jg of cytochalasin B for 5 mm at 37#{176}Cin 60 jsl. In experiments assessing the effects of sera

Scald burn injury and sham treatment of guinea pigs were performed under anesthesia as previously described [12]. The full-thickness burns covered 30% of the total body surface of the animals. The animals were resuscitated before and at 1.5 h after injury and were rested on heating blankets to reduce heat loss and minimize stress. Bacteriologic studies demonstrated that the injured animals did not develop bacteremia during the 7-day postburn period of observation.

Thermally

fluorescence

libitum.

Measurement Experimental

by

with fluorescein experiments,

for 10 mm at 37#{176}Cwith The conditions described

Male and female Hartley guinea pigs weighing 300 to 350 g were purchased from Murphy Breeding Laboratories, Plainfield, IN. The animals were housed in separate cages and adapted to the new environment for 4 to 5 days. The

Groups traperitoneal and blood

assessed

staining the PMNs mide [25]. In some

PGE2

in

the

sera.

Relationship production

between

PMNs room

10

drug

tion were

before

preincubated nonsteroidal

indomethacin

for 15 mm anti-inflamma-

or piroxicam)

or

with

above. In cytochalasin

measurement

some experiments, B under conditions of cAMP

between

data

were

‘-.3 (1) a)

the

u (0 0

0

E C

C..)

01

the PMNs of the O2

2

by analysis

of var-

Fig.

2.

after

treatment

Kinetics

treated

by PMN5 in a

Our initial studies determined O2 production in response to fMLP and PMA using PMNs obtained from injured and sham-treated animals at various time intervals during 1 week postburn. We used 10 M fMLP and 0.32 M PMA in these experiments because these concentrations were found in preliminary

experiments

to

induce

maximal

O2

production

by normal guinea pig PMNs. Responses were measured after 20 mm of incubation with the activating stimuli. O2 production by PMNs from injured animals in response to fMLP and PMA was significantly reduced during 4 days postburn and was returning to normal by 7 days postburn (P < .01; Fig. 1). O2 production was negligible in the ab-

( < 0.1 nmob

of stimulus

jured and sham-treated ing that the reduced injured animals were

per

animals responses not related

106 cells).

PMNs

from

in-

production

Sera

in

PMNs

were

response

to

pooled

serum

1 day

postburn.

with

obtained

at

are

10

20

fMLP

(A)

from

and

PMA

injured

Normal

or

(B) sham-

PMNs

Incubation did not

were

of the PMNs affect viability.

of three

separate

cx-

We next determined the kinetics of #{176}2 production and dose responses to IMLP and PMA utilizing PMNs obtained from injured and sham-treated animals at 1 day postburn. O2 production by PMNs from injured animals to both stimuli was significantly reduced during 2 through 20 mm of incubation (P < .05; data not shown). When O2 production in response to increasing concentrations of IMLP (0.1-50 M) and PMA (0.01-0.64 M) was measured, responses

with

significantly

not shown). supranormal that tered

To

the of02

normal

rum

injured

inputs

to

of

a,

guinea

from

response

pig

injured

0

.--

E

PMNs

were

to 10 zM

O2

data

are

animals

circulating

factors

obtained

at by

inhibitory activity; were significant < .05; Fig. 2A).

the

with

in-

10%

se-

for 10 mm at determined. Se1

day

normal

serum

in

thermal

incubated

animals was

al-

on O2

following

production

whereas

fMLP,

.05;

O2 production injury.

by PMNs

animals

reduced

animals had minimal tween these treatments mm of incubation (P

to

of

were