Actin

polymerization

dysfunction Sharon *Department University

contributes

following R. HassIen of Surgery, of Minnesota,

thermal

David Ramsey

to neutrophil injury

H. Ahrenholz7

Hospital,

St.

chemotactic

Lynn D. SoIem

Paul,

Minnesota

and

and Robert

Departments

D. Nelson’

of Dermatology

and

Microbiology,

Minneapolis

Abstract: The agent(s) and mechanism(s) responsible for suppression of neutrophil chemotaxis in association with major thermal injury have not been identified. We have proposed that the reduced random motility characterizing patients’ cells may contribute to their generalized

The level of polymerized actin in peripheral blood and exudate neutrophils from healthy donors and burn patients was quantified by flow cytometry of cells stained with nitrobenzoxadiazole (NBD)-phallacidin, a fluorescent reagent that binds selectively to polymerized or filamentous ac-

chemotactic dysfunction. Here we report that actin polymerization may be responsible for the loss of neutrophil motility associated with major thermal injury. Using a fluorescent ligand specific for polymerized or filamentous actin (NBD-phallacidin) in conjunction with flow cytometry, we have discovered that peripheral blood and exudate neutrophils from patients with major thermal injury contain increased levels of actin in a stably polymerized form. Because cyclic polymerization and depolymerization of actin is essential to cell motility, we suggest that actin polymerization may contribute in a major way to the attenuation of neutrophil random and chemotactic functions induced by major thermal injury. J. Leukoc. Biol. 52: 495-500; 1992.

tin [14] changes

Key

Words:

clzemotaxis

.

migration

.

F-actin

.

MATERIALS Healthy

AND

control

of

actin

conformational

16].

METHODS

and

patient

populations

Our use of human subjects for this study was approved by the Institutional Review Board at St. Paul-Ramsey Medical Center and the Committee on the Use of Human Subjects in Research at the University of Minnesota. All subjects gave signed consent to participate in this study. Thermal injury patients were adults admitted to The Burn Unit at St. PaulRamsey Medical Center. Major thermal injury was defined as 2#{176} + 3#{176} burns involving 40% of total body surface area; no further stratification based on clinical status or therapy was considered. Healthy control donors were office and laboratory personnel at the medical center.

NBD-phallacidin

INTRODUCTION Considerable progress has been made in identifying biochemical events involved in the normal chemotactic response of neutrophil leukocytes (reviewed in refs. 1 and 2). Progress has been made more slowly in identifying agents and mechanisms involved in the defective chemotactic responses of neutrophils from patients with thermal or traumatic injury, infection, or systemic disease [3]. This report describes progress we have made in studies to establish the causes of the neutrophil chemotactic dysfunction acquired in response to thermal injury. Reduced chemotaxis of peripheral blood neutrophils following thermal injury has been described in terms of number of responding cells in the membrane filter assay [4-8], distance ofcell migration in the under agarose assay [9, 10], and cell speed and orientation in a Zigmond chamber [11]. Chemotactic function ofneutrophils from patients with large

Isolation

of blood

neutrophils

Blood specimens were obtained by venipuncture using heparin as anticoagulant. A 100-jzl aliquot of each specimen was withheld as a source of neutrophils to be assayed for actin polymerization. Erythrocytes were eliminated by hypotonic lysis, and the neutrophils were washed and resuspended in phosphate-buffered saline containing 0.2% bovine serum albumin (PBS-BSA, both from Sigma Cell Culture Division, St. Louis, MO). The remaining specimen was used as a source of neutrophils prepared as pure cells by a one-step density gradient centrifugation procedure [17]. Contaminating erythrocytes were eliminated by hypotonic lysis, and the neutrophils were washed, counted, and resuspended in minimum essential medium (MEM, Sigma).

Isolation

burns becomes maximally depressed during the second week following injury and begins to normalize within another week, in the absence of complications [12]. Reduced chemotactic responses of patients’ cells to three unrelated attractants [4, 12] demonstrate that this phenomenon is not attractant specific and therefore probably not attributable exclusively to desensitization through receptor down-regulation. We have proposed that the reduced nondirected or random motility observed for burn patients’ neutrophils [12, 13] may play a part in this generalized chemotactic dysfunction. We now offer evidence that the reduced motility of patients’ neutrophils may be attributable in part to excessive stable polymerization of the actin component of the cytoskeleton.

and allows measurement by flow cytometry [15,

of exudate

neutrophils

Exudate neutrophils were isolated sites of otherwise healthy individuals sites of burn patients. The cleaned

Abbreviations:

BSA,

leucyl-phenylalanine; essential medium;

Journal

PBS, phosphate-buffered Reprint requests: 124

UMHC, Received

bovine

MCF, NBD,

Robert

University February

of Leukocyte

accidental abrasion and skin graft donor sites were covered with

fMLP,

albumin;

mean channel nitrobenzoxadiazole;

saline. D. Nelson, of

11,

serum

from

Minnesota,

1992;

Biology

accepted

Volume

fluorescence; NEM,

Department

52,

7,

MEM, minimum N-ethylmaleimide;

of Dermatology,

Minneapolis, July

N-formylmethionyl-

MN

Box

55455.

1992.

November

1992

495

To

Tegaderm (3M, St. Paul, MN) or Biobrane (Winthrop Pharmaceuticals, New York), respectively, for a period of 4 to 5 h, after which accumulated fluid was aspirated into a heparinized syringe. Cells recovered by centrifugation were washed, contaminating erythrocytes were eliminated by hypotonic lysis, and the leukocytes were resuspended in appropriate medium. Leukocytes recovered were evaluated for type and viability by examination of Wright’s-stained slides and exclusive of trypan blue dye, respectively. The exudate cells were consistently >90% mature neutrophils, unaggregated, and >95% viable. No further purification steps were performed.

Measurement Random

of random

motility

of

and

exudate

was measured using the under agarose technique [18J. Cells suspended in MEM to 5 x 10 per ml were delivered in 10-sl aliquots to wells in the agarose plates. Following incubation at 37#{176}Cfor 2 h, the cells were fixed by flooding the plates with 2.5% glutaraldehyde in phosphate-buffered saline, the agarose was removed, and the cells were stained with Wright’s stain. Random motility was measured as the distance of migration between the well margin and the leading front of cells. Distance values were expressed in centimeters from a projected image of the migration pattern magnified 40x.

of polymerized

of actin

polymerization,

we

in-

of actin of the

filament cell.

populations

RESULTS motility functions neutrophils

of peripheral

blood

and

neu-

trophils

Measurement

reversibility

to low temperature and cortical regions

Random exudate blood

the

sensitivities in lamellar

motility

peripheral

assess

cubated cells suspended in PBS-BSA at 0#{176}Cfor 30 mm before staining with NBD-phallacidin. This technique has been reported to reverse actin polymerization in chemoattractant-stimulated rabbit neutrophils to the level of filamentous actin in unstimulated cells [21]. The incomplete reversal of actin polymerization was attributed to different

actin

Actin was stained with a fluorescent analogue of phallacidin and the level of staining was quantified by flow cytometry, following the method ofHoward and Meyer [15]. Briefly, leukocytes were suspended in PBS-BSA at a concentration of 10 per ml and transferred in 0.1-ml aliquots to 5-ml polypropylene microcentrifuge tubes. The leukocytes were then fixed, permeabilized, and stained in a single step by addition of 0.1 ml of 3.7% phosphate-buffered formalin containing 100 g/ml lysophosphatidylcholine (Sigma) and 1.65 x 106 M 7-nitrobenz-2-oxa-1,3-diazole (NBD)-phallacidin (Molecular Probes, Eugene, OR). After incubation at 37#{176}Cfor 10 mm, the cells were washed once with PBS-BSA and resuspended in 0.5 ml of PBS-BSA for flow cytometric analysis of actin polymerization using a Coulter EPICS Profile instrument. Neutrophils, monocytes, and lymphocytes were discrimi-

Data from our assays ofthe random motility ofcontrols’ and patients’ peripheral blood neutrophils under agarose are summarized in Figure 1. Data used to generate this figure were derived from cells isolated from patients with major injury during the second week after injury, to illustrate the maximum influence ofthermal injury on neutrophil random motility ex vivo. Peripheral blood neutrophils isolated from 12 healthy control donors migrated 3.0 ± 0.1 cm (mean ± SEM, measured from a 40x magnification ofthe migration pattern) with a range of2.5 to 3.7 cm. Blood neutrophils isolated from eight patients during the second week following major thermal injury migrated 1.3 ± 0.6 cm with a range of 0 to 2.6 cm, representing an average 57% decrease in this motility function. Data describing the random motility functions of exudate neutrophils isolated from controls and patients are also summarized in Figure 1. Exudate neutrophils recovered from abrasion sites of four otherwise healthy individuals migrated an average 3.2 ± 0.9 cm, with a range of 1.7 to 5 cm. Exudate neutrophils recovered from skin graft donor sites of four patients with major thermal injury were consistently viable but nonmotile. Patients’ exudate neutrophils were therefore considered to provide a special opportunity to consider the mechanisms of loss of neutrophil motility.

5

4

E

nated in terms of forward versus right angle light scatter. Stained cells were excited with a 488-nm argon laser and emission was read at 530 nm with a long-pass filter. Staining by NBD-phallacidin was expressed as mean channel fluorescence on a linear scale, calculated from analysis of 10,000 neutrophils. The instrument was calibrated on a daily basis using DNAcheck (Coulter Cytometry).

‘5

>

3 0

E

2

0 -V

a: Treatments To

to polymerize

approximate

the

maximum

or depolymerize level

of actin

actin polymerization Controls Peripherei

possible in human neutrophils, we treated control neutrophils suspended in PBS with N-ethylmaleimide (NEM) for 30 mm at 37#{176}C.NEM is a penetrating sulfhydryl reagent

the advantage of neutrophil

496

of Leukocyte

Journal

gained function.

Biology

by using

phorbol

Fig. der

1. Random motility function agarose. Distance of migration

well

margin

Peripheral tion

52,

November

1992

SE

leading

total

applied to skin values

edge

neutrophils

11 control

as

derm applied

ester

to the

blood

from

tamed

±

Volume

Controls Ewdate

Source

that causes an increase in neutrophil filamentous actin [19], as well as decreases in motility and other actin-dependent neutrophil functions [20]. We selected NEM for this purpose because its effect on actin polymerization is rapid and sustamed [16, 19]. NEM has the additional advantage of bypassing surface membrane receptors to interact directly with actin, not unlike as a stimulator

Patients Blood

subjects

leukocytes

and from

to skin abrasion graft donor sites for

the

assays

cell

obtained 8 patients. aspirated

patients’ neutrophils the distance from

migration

pattern

magnified

by density

gradient

Exudate

neutrophils

fluid

accumulating

sites of four healthy of four burn patients. identified.

Fluid

of Neutrophil3

of controls’ and (cm) represents

of the

were

Patients

donors The bars

unthe 40 x.

centrifugawere under

obTega-

or Biobrane denote mean

Relationship of random polymerization

motility

dysfunction

to actin

800

I

Subsequent studies were designed to determine if the equilibrium between monomeric and polymeric actin in neutrophils might be subject to alteration by burn injury and the exudation process. Data summarized in Figure 2 describe the levels of polymerized actin in peripheral blood (A) and exudate neutrophils (B) from controls and patients. Actin polymerization is expressed in terms of mean channel fluorescence stained temperature without

(MCF, measured on a linear NBD-phallacidin without pretreatment. The fluorescence low-temperature pretreatment

scale) of cells or with lowof cells stained was taken to

with

represent

the

total

amount

of polymerized

actin

present,

the fluorescence of cells stained after low-temperature ment was considered to reflect the amount of actin reversibly polymerized. The MCF values for blood neutrophils from 11 control donors averaged

± 53 without and with cold pretreatment, respectively. Ranges for these values were 414-700 and 182-620, respectively. In percentage terms, staining of the patients’ blood neutrophils without cold treatment exceeded that of cornparable control cells by 67%. Low-temperature treatment reduced the staining of patients’ cells to only 397 channels, which exceeded the respective control value by 138%. These results demonstrate that circulating neutrophils in burn patients contain increased levels of filamentous actin, most of which appears to be irreversibly polymerized. The MCF values for exudate neutrophils from three control donors averaged 353 ± 54 and 172 ± 35 without and with cold treatment, respectively. Ranges for these control values were 278-457 and 140-243, respectively. These values demonstrate that the exudation process in healthy individuals induces only a small increase in neutrophil actin polymerization (24%) and that actin polymerization in this situation is fully reversible. The MCF values for exudate neutrophils from four patients averaged 741 ± 50 and 711 ± 30 without and with cold treatment, respectively.

A

Peripheral

BloodNeutrophils

B

800

1 :

0III 600-]

I

il ot5 is

400] 200

600-1 2001 400]

III

0 25#{176}0#{176}

Fig. 2.

Staining

a fluorescent tin.

The

MCF

level

on

neutrophils

were

pretreatment

to

peripheral

blood

(B) Staining control

values

was

scale.

the

patients’

stained

from

assays

four

actin burn

identified.

flow

with

(A)

in exudate

with

Staining

11 control patients.

cytometry

subjects

neutrophils The

bars

filamentous

and indicate

ac-

expressed

as

whether

the

(0#{176})low-temperature of

polymerized and

actin

8 burn

isolated denote

0

mean

in

patients.

from

three ±

SE

0.1

0.5

Fig.

3.

Dose

effect

peripheral

blood

cytometry

and

tions

indicate ±

NEM

expressed

SE

on The

as MCF

whether

( 0#{176}) low-temperature mean

of

neutrophils. the

values

for

NBD-phallacidin level on

of

scale.

were

stained

quantified

The

temperature

actin.

determinations

of

was

without

to depolymerize

MCF

5.0

(tM)

staining

staining

a linear

neutrophils

pretreatment

1.0

N-Eth,1rnaIeirnide

Concentration

from

flow nota-

(25#{176})or The

three

control by

replicate

bars

with

denote expert-

ments.

Ranges for these values were 539-839 and 545-750, respectively. These values demonstrate that the exudation process in patients with major injury induces a considerable increase in actin polymerization and that polymerization of actin in these cells is essentially nonreversible.

NEM treatment polymerization

of control

neutrophils

To consider how the high levels served for patients’ neutrophils are possible, we treated control

to induce

actin

of actin polymerization obare related to the levels that neutrophils from healthy

donors with NEM to induce polymerization before staining with NBD-phallacidin. Data summarized in Figure 3 describe the results of three such experiments. Maximum staining occurred following exposure of the cells to 1 M NEM for 30 mm at 37#{176}C, yielding an MCF value of 639 ± 19 (mean ± SEM). Increasing the concentration of NEM to 5 jM did not produce a further increase in staining. If the MCF value obtained for neutrophils exposed to 1 M NEM is considered to approximate a maximum level of actin polymerization, the filamentous actin content of patients’ peripheral blood neutrophils calculates to half-maximum [i.e., (478 286)/(639 286) = 54%) and actin polymerization in patients’ exudate neutrophils can be considered

burn To

NBD-phallacidin, or

notations

(25#{176}) or

actin.

neutrophils

by

temperature

without

Patients

to polymerized

quantified

The

depolymerize

and

neutrophils

0

complete maximum neutrophils completeness

25#{176}00

00

Controls

specifically

of polymerized

subjects

for

and

binding

of staining

a linear

25#{176}

200

IL

Iiiii

Petients

ofcontrols’ reagent

ExudeteNeutrophils

________________

25#{176}00

Controls

o5

treatthat is irperipheral 286 ± 14

397

-

0#{176}C

.

and

(mean ± SEM) and 167 ± 6 without and with cold treatment, respectively. Ranges for these control values were 202-360 and 147-198, respectively. The MCF values for peripheral blood neutrophils from five burn patients studied during the second week after injury averaged 478 ± 46 and

800

25#{176}C

[i.e., [741 268)/639 level of actin polymerization remains to be determined, of actin polymerization

patients. establish

the

reversibility

286)

of

= 129%]. The true inducible in control however, as does the in neutrophils from

NEM-stimulated

actin

polymerization, the neutrophils were incubated at 0#{176}Cfor 30 mm before staining with NBD-phallacidin. For cells exposed to 1 tM NEM, the low-temperature treatment reduced the average MCF value from 639 ± 19 to 210 ± 8. This temperature-induced reduction in staining was interpreted as establishing that actin polymerization stimulated by NEM, like N-formylmethionyl-leucyl-phenylalanine (fMLP)

Hasslen

et al.

[21],

is a fully

Polymerization

reversible

phenomenon

of neutrophil

actin

and

in vivo

suggest-

497

ing cur

that actin polymerization by some mechanism

after thermal duplicated by

not

injury NEM

must treatment.

oc-

DISCUSSION This

Histograms

describing

staining

of neutrophils

study

represents

an

extension

of other

investigations

of

acquired neutrophil chemotactic dysfunction conducted in our laboratory. Our earliest studies of this phenomenon involved effects of various pretreatments of isolated control neutrophils on their subsequent random and chemotactic migratory functions. We observed that cells exposed to a high dose of one chemoattractant (C5a or fMLP) exhibited a reduced chemotactic response to the other attractant in association with attenuation of random motility [22]. This observation led us to consider that loss of random motility could reduce chemotactic responses to multiple attractants without a change in the expression of surface receptors for

studied

The

histograms in Figure 4: derive from flow cytometry of NBD-phallacidin-stained neutrophils from four sources, in descending order: peripheral blood of a healthy control donor, peripheral blood of a patient with moderate thermal injury (2#{176}+ 3#{176} injury involving 20 to 40% ofbody surface area), peripheral blood of a patient with major burn injury, and exudate fluid from a skin graft donor site on a patient with major thermal injury. Cells from these sources were stained with NBD-phallacidin without or with cold treatment, as indicated. The profiles of histograms in the left

all

column establish that the cells containing increased levels of polymerized actin do not represent a discrete subpopulation of neutrophils from any of these sources. The profiles in the right column similarly establish that low-temperature treatment does not selectively affect the polymerized actin content of a discrete subpopulation of cells from these sources. The histogram pairs A-B and C-D also characterize the temperatu re-induced reversibility of actin polymerization in peripheral blood neutrophils from a healthy control subject and a patient with moderate thermal injury.

ligand

species.

The

reduced

random

and

chemotactic

migratory functions we have observed for neutrophils isolated from burn patients [12] suggested that reduced random motility might also contribute to neutrophil chemotactic dysfunction acquired in life. An inhibitory effect of cramps or muscle spasms on the pace of a marathoner toward the finish line provides an analogy of this relationship. Our subsequent studies to define the mechanism of loss of random motility exhibited by control neutrophils exposed to high-dose chemotactic factor provided evidence that autooxidative injury to the cell might be involved [23, 24]. We

PRE-TATMENT

TEMPERATURE

25#{176}C

0#{176}C

.

.

..:

C

E

Fig.

4. Histograms

phallacidin four trol

sources:

(A

subject,

tient

describing

staining (C

with

reactions

and

B) peripheral

and

D)

moderate

peripheral

blood

from

injury, and (G and with major thermal G in the left column without

B, D, from

cells

498

Each

blood

peripheral

of NBD-

blood injury,

a patient

with

#{163}

from

from

a con-

from

a pa-

(E major

and

F)

thermal

H) exudate fluid from a patient injury. Histograms A, C, E, and were derived from cells stained pretreatment.

in the

stained

pretreatment. multiple

H

spectra neutrophils

thermal

low-temperature

F, and

the for

right

column

following histogram

Histograms

were

derived

low-temperature pair

typifies

results

0

of

MEAN

CHANNELFLUOREScENcE

Journal

.

1000

NBD.-PHALLACIDIN

assays.

of Leukocyte

Biology

Volume

52,

November

1992

0

CNAHNELFLUORESCENCE

BINDING

.

1000

speculated that plasma membrane, enzyme systems, and cytoskeletal elements were all potential targets for oxidative injury affecting cell motility. A role for auto-oxidative damage as a mechanism for the neutrophil locomotory defect associated with trauma was later proposed by Maderazo et a!., together with evidence that microtubule dysfunction might be the cause of this defect [25]. The direct relationship between peroxidative microtubule dysfunction and defective neutrophil function reported by Baehner et al. [26] and Oliver et al. [27] provides additional support for microtubules as a target for oxidation-induced suppression of neutrophil motility. The actin component of the cytoskeleton has also been identified as a target of oxidative cellular injury. Hinshaw et al. reported a substantial increase in the filamentous (F-) actin content of P388D1 cell line cells exposed to hydrogen peroxide [28]. Once initiated, this change in F-actin appeared to become essentially irreversible [28] and end in shortening of microfilaments into globular aggregates [29]. Raghu et al. described similar fragmentation, disruption, and granular aggregation of actin filaments in A549 cells exposed to hydrogen peroxide [30]. Since sustained cell movement requires continuous reversible transition of monomeric to polymeric actin, an accumulation of stably polymerized actin in human neutrophils, like suppressed polymerization of actin [31-34), would be expected to translate into reduced cell motility. In this report we have described an association between increased polymerized actin content and reduced random motility of peripheral blood and exudate neutrophils of patients with major thermal injury. Patients’ peripheral blood neutrophils exhibited an average increase of 67% in staining with NBD-phallacidin in association with an average 57% reduction in motility under agarose; exudate neutrophils exhibited an average increase of 159% in staining in association with total loss of motility. Our observation that exposure to cold did not reduce the staining of these patients’ cells to control levels suggests that their polymerized actin may not be available for the reversible actin polymerization required for cell motility. It is unlikely that we did not provide sufficient time for cold-induced depolymerization to occur in the patients’ cells, since depolymerization of actin has been observed to occur in IMLP-stimulated neutrophils within 2 mm at 4#{176}C[21]. It is therefore tempting to conclude that an increase in stably polymerized actin is the cause of the reduced motility exhibited by burn patients’ neutrophils. Additional experiments are required to establish the contribution of actin polymerization to the motility dysfunction of patients’ neutrophils, but one additional consideration from our data is available to support this relationship. The random motility values for the patients’ peripheral blood neutrophils, 0 to 2.6, describe a considerable range of this

clue to this mechanism will ultimately be available from studies to define the physical state of polymerized actin in patients’ neutrophils and identify stimuli in patients’ plasma and wound fluids that reproduce this phenomenon in control neutrophils. The possibility that antibiotic and anti-inflammatory drugs might functions must also A final application contribution of actin

direct of the

alteration multiple

of actin proteins

or altered that control

function of one polymerization

neutrophil

migratory

concerns in patients’

the possible neutrophils

to cell stiffness and the influence of neutrophil stiffness on blood flow. The 8-gm diameter of neutrophils [37] is considerably larger than the 5-jim diameter ofthe smallest capillanes [38], so an ability to deform is essential for their flow through these vessels. A reduction in deformability (or increase in stiffness) would then lead to retention of neutrophils in small vessels and reduction of blood flow. A role for actin polymerization in neutrophil stiffening leading to decreased blood flow in vivo has been demonstrated by infusion of mice with fMLP-stimulated neutrophils [39]. Neutrophils stiffened by stable actin polymerization may similarly plug small capillaries in burn patients; limit delivery of leukocytes, oxygen, and nutrients to wound sites; and compromise both host defense functions and wound healing. Adjunctive therapy for burn injury may therefore ultimately indude agents that prevent actin polymerization or promote its depolymerization, e.g., pentoxyfylline [40, 41].

ACKNOWLEDGMENT This project was supported in part by a grant from the International Association of Fire Fighters Burn Foundation (R.D.N.), grant ROl Al 22374 from the National Institutes ofHealth (R.D.N.), and grants from the Ramsey Foundation (L.D.S.).

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function. Likewise, the MCF values from staining with NBD-phallacidin without cold pretreatment, 341 to 577, represent a range of polymerized actin content. Thus, regression analysis of these data can provide a test of their functional relationship. The calculated r2 value of 0.8 suggests that loss of random motility of patients’ circulating neutrophils is primarily attributable to actin polymerization. Q uestions remaining to be answered are related to the stimulus involved and the mechanism of stable actin polymerization. Stimuli must include the complement components C5a and CSadesArg [10, 13, 35], but other agents are probably also involved in either agonist or priming roles. The mechanism of stable actin polymerization may involve

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