~
WILLlAM BROWDER, M.D., DAVID WILLlAMS, PH.D., HENRY PRETUS, PH.D., GIORGIO OLlVERO, M.D.,
FRANCESCO ENRICHENS, M.D., PATRIZIO MAO, M.D., and ALLESSANDRO FRANCHELLO, M.D.
Host immunosuppressionafter trauma contributes to septic
morbidity. The macrophageis a key elementin the host immune
response.This studyevaluatedglucan,a macrophagestimulant,
in a prospective,randomized,double-blind study of 38 trauma
patients undergoingsurgery. Glucan (21 patients), 50 mg/m2,
or placebo(17 patients),,'as givenintravenouslydaily for 7 days.
Delayed hypersensiti\ity skin testing was performed on days1
and 7 after trauma. Serum interleukin-1 (IL-I) and tumor necrasis factor (TNF) ,,'ere assayedafter trauma. While the total
mortality rate ,,'as significantly less in the glucan group (0%
versus29%) (p < 0.05), the mortality rafe from sepsis,,'as not
statistically different (0% versus17.6%).Glucan therapy significantly decreasedseptic morbidity (9.5% versus49%; p < 0.05).
Serum IL-I had a greater increase in glucanpatients on dar 3
after trauma (143.4 :!: 19.3% versus78.6 :!: 11.7%; p < 0.05),
but there ,,'asno differencethereafter. SerumTNF did not vary
bet,,'eengroups. Early increase in IL-I correlated with subsequent skin test com'ersionto positive. Neither serum IL-I nor
TNF "'as a reliable indicator of roture sepsis. Further clinical
trials are indicated to e\'aluatebiologic responsemodifiers that
activate macrophagesin the trauma patient.
T
HE PASTDECADE
has witnessedmajar advances
in the care ofthe trauma patient. Improved field
resuscitationand rapid transportto levell trauma
centers have resulted in improved early survival after
traumatic injury. However subsequentmorbidity and
mortality related to sepsisare still majar problems in the
complete recoveryofthe trauma patient. A primary factor
in the developmentofsepsisis depressionofhost-immune
responseafter severeinjury. Previous studies have documented that both cellularl-3 and humoral4-6immunity
are diminished in the patient with severeinjury. Altered
macrophagefunction is reported to be a key element in
the increasedsusceptibilityto sepsisafter trauma!.8 An-
From the Departments of Surgery and Physiology, Tulane
University School of Medicine, New Orleans, Louisiana; and
Istituto Di Chirurgia D'Urgenza, University
of Torino, Torino, Italy
tigen processingand presentation by the macrophage,
which is essentialfor proper lymphocyte response,is suppressedafter hemorrhage7and tissue trauma.8 Macrophagesecretoryproducts suchas interleukin-l (IL-l) and
interferon, which are important mediators of immune
defense, are decreased after trauma.9 In response to
trauma, so-calledinhibitory macrophagesproduce prostaglandin E2 (PGE2),which is capable of suppressinga
varietyofimmune functions.1oIn addition serumopsonin
levels and reticuloendothelial clearance of bacteria are
depressedin the trauma patient.ll
Given the central role ofthe macrophagein mediating
the overall immune response,a reasonableapproach to
the amelioration of immune suppressionin the trauma
patient might include biologic responsemodifiers (BRM)
that enhance macrophagefunction. Our laboratory has
extensivelystudiedglucan,a potent macrophagestimulant
that has been shownto be beneficial in animal models of
sepsis,12,13
trauma,14and wound healing.15The present
study wasdesignedto assessthe value of glucan in a prospective trial of trauma patients. In addition the monokines IL-I and tumor necrosisfactor (TNF), which may
be important in maintaining host-immune responsiveness,16-18
were measuredin the early post-trauma period,
and correlated with delayed hypersensitivity skin testing
and the subsequentdevelopment of sepsis.
Methods
Presentedat the 10lst Annual Meeting ofthe Southern SurgicalAssociation, Hot Springs,Virginia, December3-6, 1989.
Addressreprint requeststo Dr. William Browder, Tulane University
Department ofSurgery, 1430Tulane Avenue, New Orleans,LA 70112.
Accepted for publication January 19, 1990.
Palienl Populalion
The Trauma Institute in Torino, Italy is the equivalent
of a level l trauma center and is the only such facility
605
606
BROWDER AND OTHERS
serying approximately 1.2 million people.The study was
designedto evaluatepatientsadmitted to the Trauma Institute betweenthe ages18 and 65 yearswho underwent
exploratory laparotomy or thoracotomy for trauma. Excluded patientsincluded thosewith cirrhosis,renal failure,
severehead injury, or reproductive potential. Eligible patients were enteredjoto a prospective,randomized,double-blind study to receive either glucan(50mgtm1 or saline placebointravenouslydaily for 7 daysafteroperation.
This dose of glucan has been demonstratedto increase
interleukin-l and interleukin-2 production in humans.19
All colon injuries weretreatedwith colostomyand delayed
primary wound closure.AII patientsreceivedprophylactic
antibiotics, usuallya cephalosporin,for 5 to 7 days.Subsequent antibiotic therapy was contingent on signs of
clinical infection, as well as appropriate culture and sensitivity results. Patients underwent intraoperative autotransfusion as indicated. Parenteralnutrition was begun
immediately after operation in all patientsand continued
until bowel function returned. Postoperativeventilator
support was used in all patients. Weaning from the ventilator and subsequentextubation was scheduledon an
individual patient basis. An index of injury severitywas
established by calculating the Injury Severity Score
(ISSfo,21Clndthe Abdominal Trauma Index,22as previously described.
Skin tests were applied on days 1 and 7 after trauma
using the Multi- Test device,23which usessevenantigens
(tetanus, diphtheria, streptokinase/dornase,tuburculin,
candidin, tricophytin, and proteus) and one control.
Anergy wasjudged to be lessiban two reactionsof 2 mm
induration.24
A clinical infection wasdocumentedwhenthe patient's
temperatureraseabove39 C daily and there wasevidence
of majar soft-tissue involvement with positive bacterial
cultures (wound, lung, or abdomen).
This clinical protocol was approved by both the Committee on Use of Human Subjectsat Tulane University
and the Institutional Review Board ofthe Trauma Institute, University of Torino, Torino, Italy. Informed consent was obtained from all patients or their immediate
next-of-kin.
Glucan Preparation
Soluble glucan was prepared in the Departments of
Surgeryand Physiologylaboratories, Tulane University
School of Medicine. Particulate glucan was preparedby
a modification ora previouslydescribedprocedure!5 Soluble glucan was prepared according to the processdesct:ibed in United States Patent 4,739,046. The glucan
was shown to be endotoxin free as determined by
the Limulus Iysate procedure (Sigma Chemical Co., Sto
Louis, MO).
Ann. Sur¡.. May 1990
BloodAnalysis
Blood was'collectedin siliconized giasstubes on days
1,3, 5, 7, and 9 after trauma for determination of serum
IL-I levels. Similarly blood wascollected on days 2, 4, 6,
8, and 10 for tumor necrosis factor assay. Leukocyte
counts were determined daily for the first week after operative intervention. Serum for IL-I .Band TNF-a determinations wasstored frozen in plastic vials until assayed.
Radioimmunoassayo/ IL-l .8 and TNF-a
Serum levels of IL-1 fJ or TNF-a were determined by
radioimmunoassayusingcommercial1yavailablekits (1251IL- 1, RIA kit, AdvancedMagnetics,Cambridge,MA and
1251-TNF-TEST,
Genzyme Corp., Boston, MA, respectively). For either assay,a IOO-JLLaliquot ofunextracted
serumwas assayeddirectly, and al1sampleswere ron in
duplicate. Cross reactivity for the antibody included in
each kit was negligible for other cytokines.
StatisticalAnalysis
Statistical comparisons were performed by one-way
analysis of variance where appropriate. Differences between groups were determined using a Bonferroni multiple comparisontest. Additional comparisonswere conducted using chi-square with Yates correction and the
Student's t test.
Statistical significancewas assumedat p < 0.05. Statistical studies were performed using STAT A Statistical
SoftwarePackage(Computing ResourceCenter, Los Angeles,CA) ron on an IBM-compatible personalcomputer.
Results
From 1986to 1988,a total of 38 patientswere entered
in the study (21 glucan, 17placebo).There were 19 male
and 2 female patients in the glucan group and 12 male
and 5 femalepatients in the placebogroup. Blunt trauma
due to automobile accidentwasthe most common mode
of injury, accountingfor 12of the 21 injuries in the glucan
group and 15 of the 17 injuries in the placebo group.
Penetrating injuries due to gunshot or stab wounds accounted for the majority ofthe remaining patients.While
the glucan-treatedpatients had injuries to the lung and
colon, the placebo group had more liver and duodenal
injuries. Howeverthere wasno statistically significantdifference in the distribution of organ injuries betweenthe
two groups (Table 1).
A comparison of injury severity in the two groups of
patientsis listed in Table 2. Injury severityand ATI scores
weresimilar. In addition therewasno significantdifference
betweenthe two groups in the amount of blood transfusion perioperatively or in the number of patients pre-
TABLE Breakdoll'n alargan
Injury in 38 Trauma Pafienfs Admilfed
lo lhe Clinical PrOloco/"
Organ Injured
Placebo
Glucan
8
4
5
2
3
I
3
I
II~-':;<'
607
MACROPHAGE ACTIVA TION IN TRAUMA
VoI.211.No.S
7
6
5
O
T ABLE 3. Description o/ Septic Morbidity in Trauma Patients
Receiving Placebo or Glucan Treatment
PlaceboPatients Glucan Patients p Value
Complication
Abdominal abscess
Pneumonia
Wound infection
Generalizedsepsis
Total
4
o
2
2
O
1
O
8
2
nos.
nos.
n.s.
n.s.
<0.05
o
.There was no significant variation betweenplaceboand glucan patients with regardto organ injury.
senting to the emergencydepartment with systolicblood
pressurelessthan 90 mm Hg.
Delayed H)persensitivity
In this protocol, patients who were anergic on dar 7,
regardlessof treatment group, had a greaterincidence of
subsequentsepsisthan reactive patients (53.3% versus
8.7%; p < 0.0 1). A total of 14 placebo patients and 15
gIucanpatientsshowednegativeskin testingon dar 1 after
surgery. However the gIucan-treatedpatients had a statistically significantincreasein conversionto positive skin
testing on dar 7 when compared to the placebo group
(73% versus21%; p < 0.02).
Morbidity and Mortality
Neither the ISS nor the A TI correlated with development of sepsis.Howeverthosepatients developingsepsis
did receive a greateramount of blood transfusion when
comparedto thosewho did not havesepticcomplications
(1333.3 :t 795.3 cc versus934.4 :t 398.4cc; P < 0.05).
Serum IL-I
fJ and Serum TNF a Assays
The percentageincreasein serumIL-llevels for glucan
and placebo patients is shown in Figure l. The glucan
patients had a significant increase in IL-l on day three
(143.4%::t 19.3% versus78.6%::t 11.7%;p < 0.05). By
day five and thereafter,there was no differencein the IL1 levels betweenthe two groups.The earlyincreasein IL1 in glucan patients correlated with subsequentskin test
conversion on day 7 (Fig. 2). Glucan patients who converted to positive skin testing had a significantly greater
increase in IL-l levels (166.8% ::t 12.4%versus 119.8%
::t 4.2%; p < 0.02).
There was no difference in the serum TNF levels of
placebo- or glucan-treated'patients (Fig. 3). In addition
there was no correlation betweenthe serum IL-l or TNF
levels alone and the subsequentdevelopment of sepsis,
regardlessofplacebo or glucan therapy (Fig. 4).
Total mortality wassignificantlylessin the glucangroup
when compared to placebo (0% versus29%; p < 0.05).
Howevertwo ofthe placebodeathswerecardiacarrhythmias secondaryto a pulmonary embolusand myocardial
infarction. The three placebodeathsdue to sepsis(17.6%)
were not significantly greateriban the zero mortality in
the glucan group. The three placebo deaths from sepsis
were secondaryto abdominal abscess(2) and pneumonia (1).
Morbidity from sepsiswas significantly greaterin the
placebo group (49% versus9.5%; p < 0.05). Specificmorbidity is shown in Table 3. The four abdominal abscesses
>,
were secondaryto duodenal (2), hepatic (1), or bowel in~
O
juries (1), and allweré1reatéd with reoperation, with two
O
subsequentdeaths.
.p
( 0.05 n
placebo
175150-
/
125
100
75-
.""""
1
~
TABlE 2. lnjury
Severity in Glucan and Placebo Patients
PlaceboPatients
Glucan Patients
ISS.
35.7 years
32.9years
23 (8-34)
ATIt
Admission hypotension
(BP < 90 mm/Hg)
Amount ofblood transfusion
26 ( 16-41)
14.4( 0-35)
41% (7/17)
5.3 units
Factor
Age
.Injury SeverityScore.
t Abdominal Trauma Index,
15.8 (8-26)
28.6%(6/21)
5.0units
5025.
O!O
1
I
admiasion
2
3
4
5
o-o
Placebo
e-e
Glucan
6
7
8
TIME (days)
F1G.l. Elevatedserum IL-I (3levels in glucan-treatedtrauma patients.
Serumwas obtained from placeboand glucan patients on days 1, 3, 5,
and 7 after trauma. IL-I (3 was quantitated by radioimmunoassay. A
significant increasein IL-I (3was observedin glucan-treatedtrauma patients on day 3.
~
608
BROWDER AND OTHERS
Ann. SUI¡. .May
1990
200:
"",
I -
175J
150.
'::;' 150.1
...J >e
G
Q
~ -o
'Qj'-'~
cn
>.
G
100-
A
50.
~
125-
"O 100:.
75J
50.
I
25~
0Anergic
Serum IL-l
Conversion
Skin Test Conversion
AG. 2. Positive correlation between elevated serum IL-1 {J levels and
skin test conversion in trauma patients treated with glucan. The increase
in IL-1 {J was significantly greater (p < 0.05) in glucan-treated trauma
patients who con verted as opposed to those who remained anergic. Ski n
testing was undertaken on days I and 7 ~ith a Multi- Test device using
seven antigens.
LeukocyteCount
Peripheralleukocyte counts for the two groupsduring
the first week after operation are presentedin Figure 5.
While the mean white blood cell counts in the glucan.
treated patientswere higher on eachofthe days,the wide
variation precluded statisticalsignificancein ibis study.
Discussion
Severetrauma has a major impact on homeostasis.In
addition to derangementsin endocrine and metabolic
functions, there is severedepressionof host-immune response.Severa!studieshavedocumentedsuchsuppression
occurring in both humoral and cell-mediated immune
responses.I,26,27
Lymphocyte activity, asmeasuredby mitogenresponsiveness,
is decreased.1In addition there is a
P
AG. 4. Lack of correlation betweenserumIL-! .Bor TNFa and deve!opment of septic morbidity in trauma patients.IL-! .Band TNFa were
quantitated by radioimmunoassay.
relative increasein the subsetof suppressorlymphocy1es.1
Polymorphonuclearleukocy1e(PMN) function is alsodepressed,as denoted by decreasedchemotaxis, phagocytosis, and intracellular killing in PMNs isolated from the
trauma patient!6,27
Humoral immunity is also altered, as retlected by reduced serum immunoglobulins after severetrauma.4,28
Similarly altered nonspecificimmunity in the form of fibronectin-mediated clearance of blood-born particulate
matter by the reticuloendothelial systemhas beenimplicated in the pathogenesisof sepsisafter severetrauma.29
Specificallydepleted fibronectin and subsequentdi minished blood clearancehas led to multiple-organ failure
and sepsisin the severelyinjured patient.29,30
Recent years have seenthe macrophageemergeas a
key cell in immune dynamics after trauma. Macrophage
production of cy1okinesthat participate in virtually all
aspectsof cellular and humoral immunity make it an important mediatorofhost-immune response.Thereforeany
alteration in macrophagefunction can significantly affect
o
1
2
3
Time (days alter
FIG. 3. Serum TNFa levels in trauma patients after placero or glucan
treatment. Serum was collected on days 2, 4, 6, and 8 after trauma.
TNFa was quantitated by radioimmunoassay.There was no significant
differencebetweentreatment groups.
Serum TNFa
4
5
6
admission)
AG. 5. Daily peripheralleukoc}1ecountsin trauma patientsafter placebo
or glucantreatment. There was no significant differencebetweentreatment groups.
Vol.211.No.5
MACROPHAGEACTIVATION IN TRAUMA
609
iromunologic responsiveness. An intriguing concept decreased, contributing to improved immune responsiveness.40
scribes two general classes of macrophages: facilitory and
In the present study, glucan-treated patients had a siginhibitory?6.31.32Facilitory macrophages are noted for the
nificant increase in IL-l levels on day 3 "after trauma.
presence of la antigen and the production of stimulatory
monokines that interact with T -helper lymphocytes. InHowever by day 5 and thereafter, there was no difference
in IL-llevels between t:hetwo groups. The immune status
hibitory macrophages, on the other hand, are recognized
of the trauma patient in this early period may be critical
lI>ytheir lack orla antigen and production ofprostaglandin
for subsequent development of sepsis. Faist et al.lo reE2 (PGE2), which interacts with T -suppressor lympho~ytes.1O.26.31.32
In particular the production of PGE2 by
ported suppressed in vitro mononuclear cell function on
days 5 to 7 after trauma. This depression correlated with
inhibitory macrophages may represent an important immunosuppressive mechanism because these cells are indevelopment of subsequent infectious complications.JO
Similarly,
Rodrick et al.9 studied mononuclear cells from
~reased after traumatic injury.33,34Among PGE2s manyo
trauma patients after operation. There was a significant
immunosuppressive effects, augmentation ofT -suppressor
decrease in IL-l production from days 1 through 5 after
tell proliferation, and inhibition of T -helper and B-cell
trauma, with a subsequent return to the normal range.9
proliferation have been well documented.33 In addition
Stephan et al.s used a murine model to study membrane
PGE2 inhibits production of certain cytokines such as ILIL-l activity of peritoneal macrophages after laparotomy.
1 and IL-2, which are important for proper T -lymphocyte
They noted a marked depression of IL-l activity on days
response.33It is apparent that PGE2 release by inhibitory
1 and 3 after laparotomy, with activity returning to normal
tnacrophages may playa major role in post-traumatic imby day 5 after trauma.s It is apparent that macrophage
~unosuppression. Several animal studies have docudepression early in the period following severe trauma
rnented the beneficial effects of ibuprofen, a cycloxygenase
may be a majar factor in subsequent development of sepinhibitor, in models of traumatic and postburn sepsis.
sisoIt is encouraging that in these studies IL-l production
lnhibition of prostaglandin synthesis by ibuprofen in these
.was significantly greater after glucan therapy i~ the early
rnodels resulted in increased survival.3s,36
post-trauma periodo Why glucan therapy was unable to
A possible therapeutic approach to post-traumatic imenhance IL-llevels beyond day 3 after trauma is unclear.
rnunosuppression might involve the concept of selective
However this may relate to the dose of glucan used in
increase and enhancement of facilitory macrophages.
these patients. Further studies are necessary to better dePrevious studies have demonstrated that glucan, a betalineate dose-response curves for glucan therapy.
l, 3-linked glucopyranose polymer, isolated from the inner
Those glucan patients whocconverted from a negative
cell wall of Saccharomyces cerevisiae, is a potent macskin test on day 1 to a positÍve skin test on day 7 after
rophage stimulant and is beneficial in the therapy of experimental bacterial, viral, and fungal diseases.12,13,37,38trauma had a significantly increased IL-l level when
compared to the glucan patients who did not convert.
Use of glucan in a murine model ofhind-limb crush injury
The macrophage is a key cell in delayed hypersensitivity
decreased macrophage PGE2 release while stimulating
and the positive correlation between IL-llevels and skinbone marrow proliferation.14 This reversal ofmacrophage
test reactivity further supports the concept of macrophage
function toward the facilitory state might be of majar
~ctivation in these patients. Skin test results also correlated
benefit to the trauma patient. In addition glucan is water
with development of subsequent septic complications, in
soluble and nontoxic and is thus suitable for parenteral
agreement with many previous studies.42-44However in
administration in humans.39
the present study measurement of serum IL-l alone did
As noted previously macrophage production of IL-I is
an indicator ofthe activated state ofthe macrophage.13,IS
Several studies have demonstrated that macrophage act.ivation by glucan results in a marked increase in IL-I
prüduction.13,IS Intravenous glucan administration in
humans has likewise resulted in pronounced increases in
IL-l and IL-2.19 In addition to reflecting the functional
~tatus of the macrophage, IL-l may exert protective effects
in the trauma patient. IL-l activates lymphoc)l1eS and
'promotes lymphocyte release of IL-2, with resulting enhanced lymphocyte number and function.4O IL-l also enhances mobilization of polymorphonuclear leukocytes
from bone marrow and enhances their chemotactic ability.41 IL-l triggers the acute-phase response in which hepatic synthesis of certain proteins is dramatically in-
predict subsequent septic complications.
This prospective randomized study resulted in very
comparable study groups. While there was some variability inorgan injuries, overall trauma severity as assessed
by ISS and ATI was very similar. In addition there was
no significant difference between groups in the incidence
ofhypotension or the amount ofblood transfused. While
overall m ortal it y was significantly decreased in the glucantreated patients, two ofthe five deaths in the placebo group
were cardiac in nature and unrelated to sepsis. The three
deaths from sepsis in the placebo group (versus Done in
the glucan-treated patients), while not statistically significant, certainly suggest that enhanced macrophage function imparts some protection to the trauma patient. The
---,,"!~
610
BROWOER ANO OTHERS
overall incidence of sepsis was significantly greater in the
placebo group. More significant were the four abdominal
abscessesnoted in the placero group versus Done in the
gIucan-treated patients. Two ofthe three septic deaths in
the placero group resulted from multiple end organ failure
after persistent abdominal sepsisunresponsive to surgical
drainage and antibiotic therapy. Similar results have been
reported by Meakins et al.,45 who performed a doubleblind study in surgicaI patients using levamisole to prevent
infection in those high-risk patients who were anergic or
relatively anergic to delayed hypersensitivity skin testing.
While overall mortality was not different in the two
groups, levamisole recipients did haye significantly fewer
septic complications. This clinical success was accompanied by improved neutrophil chemotaxis and skin. test
reactivity.45
Previous studies have demonstrated that systemic admil1istration of gIucan mar impart protective effects in
the peritoneaI cavity.12,13,46
In animal models of Escherichia coli peritonitis, gIucan therapy increased the number and function of macrophages in the peritoneal cavity.12,13In addition PMN number and function were increased peripheraIly as well as in the peritoneal cavity.12.46
Thus gIucan therapy in the immediate post-traumatic period mar mobilize monocyte and PMN celllines to enhance antimicrobiaI defenses in the peritoneal cavity.
Recent studies have focused on TNF and its role as a
mediator in a variety of pathologic states.47.48There is
good evidence that TNF produces the metabolic, cardiovascular, and inflammatory changes seen with endotoxin.
Tumor necrosis factor mar represent an essentiaI mediator
in the complex pathophysiology of endotoxic shock.47.48
However there is evidence that TNF mar impart some
protection in septic patients. Tumor necrosis factor has
been documented to increase both polymorphonuclear
leukocyte and monocyte cytotoxicityl7,49 and mar influence release of other cytokines, especially IL-l and granulocyte macrophage-colony
stimulating factor (GMCSF).16,50Livingston et al.16have studied TNF in combination with antibiotics in a murine model of infection
following hemorrhagic shock. While TNF alone did not
reduce infection, its addition to the antibiotic regimen
significantly improved results when compared to the antibiotic alone.16 It is obvious that the ultimate effect of
TNF on the host is dependent on both the dose of TNF
and the interaction ofTNF with other mediators present
in the biologic milieu. In the current study, gIucan therapy
did not result in an increased level of serum TNF. Furthermore serum TNF did not correlate with subsequent
development of sepsis. Further studies are needed to better
delineate the role of TNF in the development of posttraumatic sepsis.
-The amount of perioperative blood transfusion in the
two treatment groups was the same in this study. However
Ann. Surg.. May 1990
those patients who subsequently developed sepsisreceived
significantly more transfused blood than did those patients
who had no séptic morbidity. This is not surprising because there is increasing evidence that blood transfusion
is immunosuppressive.51.52 Many studies have documented that the macrophage may be a key factor in
this post-transfusion immunosuppression.52-54Decreased
lymphocyteresponsiveness in post-transfusion patients
has been related to impaired macrophage lymphocyte interactions, possibly mediated by prostaglandin E2 released
from the inhibitory macrophage.52.53Macrophage chemotaxis and phagocytosis are also impaired after blood
transfusions.53 Severalstudies have linked post-transfusion
immunosuppression to increased susceptibility to infection.54.55Wound innoculation with Pseudomonas aeruginosa in a rat bum model resulted in higher mortality
Tales when rats were resuscitated with allogeneic blood as
opposed to saline or syngeneic blood transfusion.54 It has
also been demonstrated that survival in a rat E. coli peritonitis model was significantly reduced after allogeneic
blood transfusion.55 It is apparent that biologic response
modifiers that enhance macrophage function may playa
Tole in ameliorating or preventing immunosuppression
after blood transfusion. Findings in this study support
that concepto
A potential protective mechanism with glucan stimulation is macrophage release of GM-CSF. This phenomenon has striking effects on boDe marrow proliferation.
Patchen et al.56,5?
have demonstrated that glucan therapy
will significantly accelerate hematopoetic recovery in both
sublethally and lethally irradiated mice, even when the
glucan was administered after irradiation. Glucan therapy
in a mouse hind limb crush injury model caused a significant increase in boDe marrow proliferation when
compared to placebo therapy.14 While boDe marrow proliferation was not assayed directly in the present study, it
is of interest that the glucan patients in the first week after
trauma had consistently greater leukocyte counts than the
placebo group, although the differences were not statistically significant. Ready availability of peripheral leukocytes obviously would be a positive factor in resistance
to development of sepsis.
Glucan has been shown to exert many, ifnot all, ofits
protective effects vio macrophage participation.12-14 The
precise mechanism by which glucan activates macrophages is poorly understood. However Czop and Usten58
and Goldman59 have demonstrated the presence of specific
beta- 1, 3-glucan receptors on human macrophages and
mast cell liDes. Activation of the befa glucan receptors
may be the signal that initiates the cascade ofbiochemical
events that culminate in macrophage activation. Studies
are underway to better delineate this glucan receptor
mechanism.
In summary to assessthe use ofbiologic response mod-
MACROPHAGE ACfIVATION
Vol. 211oNo. S
ifiers that enhancemacrophagefunction, glucan,a beta1,3 polyglucosewas used in a prospective,double-blind,
randomized study oftrauma patients.While glucantherapy did not significantly decreasemortality ratesdue to
sepsis,the overall rate of sepsiswas less in the glucantreated patients. Glucan-treated patients had a significantly increasedserum IL-l level on day 3 after trauma.
However by day 5 and thereafter,there wasno difference
in serum IL-I levels betweengroups.This early increase
in IL-I correlated with subsequentskin test conversion
from anergyto a positive response.Glucan therapy haQ
no effecton serumTNF levels.Furthennore neitherserum
IL-I nor serum TNF levelswerepredictive of subsequent
septiccomplications. Basedon thesepreliminary results,
we believe further clinical trials with BRMs that enhance
macrophagefunction are indicated in trauma patients.
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3. Keane RM, Birmingham W, ShatneyCM, et al. Prediction of sepsis
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4. Hershman MJ, CheadleWG, George CD, et al. The responseof
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acti\1tyasa resultofthermal
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9. Rodrick ML, Wood JJ, O'Mahony JB, et al. Mechanisms of immunosuppressionassociatedwith severenonthermal traumatic
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DISCUSSJON
DR. CHRISTOPHERBAKER (Chapel HiII, North Carolina): Dr. Browder
has contributed extensively to the literature on glucans, the polysacharide
component of zymosan, as an immunomodulator, and is the major, if
not the only, surgicaI contributor in this area studying pancreatitis, wound
healing, and peritonitis in the last few yea~.
Much of the clinical work on immunomodulato~
in humans has
been done in Europe for various reasons; for instance, thymopentin in
Belgium, NSAIDS in Germany "ith Faist (a collaborator ofmine), and
now Dr. Browder's study in ltaiy. Based on the work presented and the
material in the manuscript, I have some methodologic questions and a
couple comments.
Fi~t, how did you select the patients? One of the problems in these
studies has aIways been trying to stratif)' the patients at risk, and I wonder
if you could elaborate a little bit more.
I was interested in the use ofprophylactic antibiotics for 5 days because
that is generally not my practice. Could you comment on that?
Can you comment on how glucan convens the anergic patients? Skjn
testing is obviously a very complex afea. I notice that you had a large
number of negative patients the fi~t day after operation, which I aIways
thought was due to edema, but nobody has ever written much about it.
In the data that you showed here this afternoon, you mentioned the
correlation of IL-I and conve~ion on dar 3, but the other data for skjn
test conve~ions were on dar 7 when the IL-I levels were not different.
Perhaps you can comment on that,
Were the four abdominal abscessesin the placebo group due to late
recognition of injuries or were they abscessesafter an operation for the
injury?
As a methodologic point, you expressed the IL-I data as a percentage
ofthe dar 1 levels, which is a technique that we had to use a number of
yea~ ago because patients have a "ide variation between individuals in
terms of the level of response. Did you see this in terms of the raw
numbe~, and was that why you expressed the data as percentage? Ifthis
is so, why did you express the TNF data in picograms instead of as a
percentage? It mar be that the TNF data would also be significant ifthey
were analyzed in the same way. We have usually followed patients in
human studies for 3 weeks, and I wondered why you only followed them
for 10 days,
I wonder if you thought about measuring the presence or absence of
la antigen on the surface of these macrophages? As you know, other~
Ann.Sur¡.. M.y 1990
52. Lenhard V, Maassen G, Grosse- Wilde H, et al. Effect ofblood transfusion on immunoregulatory mononuclear cells in prospective
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modelo Ann Surg 1986; 204:681-685.
54. Waymack JP, Robb E, Alexander JW. Effect of transfusion on immune function in a traumatized animal model.lI. Effect on mortality rate following septic challenge. Arch Surg 1987; 122:935939.
55. Waymack JP, Warden GD, Alexander JW, et al. Effect of blood
transfusion and anesthesia on resistence to bacterial peritonitis.
J Surg Res 1987; 42:528-535.
56. Patchen ML, DiLuzio NR, Jacques P, et al. Soluble polYgIycans
enhance recovery from cobalt-6O-induced
hemopoietic injury.
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57. Patchen ML, D' Alesandro MM, Brook 1, et al. Glucan: mechanisms
involved in its radioprotective effect. J Leukoc)'te Biology 1987;
42:95-105.
58. Czop JK, Austen KF. A beta gIucan inhibitable receptor on human
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134:2588-2593.
59. Goldman R. Induction ora beta-l, 3-D-gIucan receptor in P388D
I cells treated mth retinoic acid or 1, 25-<iihydroxY\ltarnin D3.
Immunology 1988; 63:318-324.
investigators have suggested that la allows us to separate or differentiate
faci1itory macrophages from inhibitory macrophages, and this might alIow
you to stratif)' responsiveness or at least understand future clinical studies.
Finally, I have a question arising out ofignorance. Because glucan is
a breakdo\\-n or one afilie components ofzymosan, and given the material that Dr. Deitch showed us earlier, did you see any gut-related
complications in these patients?
DR. JORGE RODRIGUEZ (Buffalo, New York): This paper is a prospective, double-blind, randomized study. It clearly demonstrates that a
biologic response modifier, glucan, increases macrophage monokine
production in association with conversion for skin testing and a decrease
in the overall septic response or rateo
My Questions are centered on the study design, the study population,
and associated macrophage function.
The criteria for the inclusion ofthe study appears to be based solely
on operative procedure. Was there any attempt to develop an inclusion
criteria based on the level of severity of injury or contamination at the
time of injuf)1
Did the use of prophylactic anbibiotics, as mentioned by Dr. Baker,
for 5 to 7 days without certification for contamination influence your
rate of septic complication?
Did the use of mechanical venti!ation on all postoperative patients
influence your pulmonary infection raje?
Thirty-eight per cent ofthe placebo group, with the higher septic rate,
versus 6 percent ofthe study population, with the lower septic rate, were
involved in penetrating injury. Could ibis, in fact, account for the difference in the septic rate?
Did the increase in macrophage production of monokine correlate
with any measure of antigen-processing presentation or a decrease in
PGE-2 production?
My last Question concems the minimal increase in serum T:--"F in both
groups. Could this be correlated to the time of specimen collection?
DR. WILLIAM BROWDER (Oosing discussion): Dr. Baker brought up
the selection of patients. Because of the requirernents for proper consent
and Hurnan Use Cornrnittee approval, we had to exclude the patients I
Jisted on the slide, narnely, those with pre-existing renal or hepatic disease
and patients with severe head injury.
Serious head injury seerned to be the injury that disqualified the rnost
Vol.211-No.S
MACROPHAGE AcnVATION
patients, because this trauma center sees quite a number of multiply
injured patients. In addition, we tried to restrict the study to patients
who underwent operative intervention, again trying to document a specific
injury as opposed to blunt trauma or pelvic fracture patients who did
not undergo operative intervention. In these Jatter patients we would
never be able to specify the exact extent ofthe injury.
The 5 days of antibiotics caused some concern. This is the standard
policy at the Trauma Institute where the study was carried out. At least
I can say that it was consistent throughout both groups of patients.
Dr. Baker asked about the raje of the macrophage in the skin test
conversion. Unfortunately we did not look specifically at that. In this
study we might hypothesize that macrophage antigen processing and
presentation would be an essential aspect of proper lymphoctye res'ponse
and positive skin test reactivity. There have been many studies showing
that this macrophage antigen processing ability is lost with hemorrhage
and hemorrhagic shock and trauma. Thus this is a possibility, although
we have no direct evidence in this study.
AII of these abdominal absoessesfollowed operative intervention. There
were two after duodenal injuries, one after hepatic injury, and one after
a bowel injury.
Dr. Baker brought up the point about expressing IL-I values as a
percentage increase, and he was correcto Because of the wide variability
of ILol in these patients, it is very difficult to get any meaningful raw
numbers.
The same was true for the serum TNF levels. If you looked at the
percentage increase in that group, they showed no difference between
the glucan or placebo groups.
The IO-day limit on the study, as opposed to 3 or 4 weeks, was determined simply because of availablity of the radioimmunoassay. Certainly the next step in this area ofinvestigation would be to follow these
patients longer as they develop septic complications, and to look at what
immune parameters are disrupted at that time.
The lA antigen on the macrophage was mentioned. We did not study
this factor. Again, we preferred to use interleukinol as the marker for
reflecting facilitory macrophages or macrophage activation.
The question ofzymosan versus glucan is an intersting one. Historically,
I
IN TRAUMA
613
Nick DiLuzio, my late mentor in New Orlean's,was proud of the fact
that he wasthe firstto showthat glucanwas thepure polYglucoseelement
of zymosan,that is, zymosandevoid of the protein moieties and other
impurities. He believedvery strongly that they were two different compounds, with glucanpossessingthe immunostimulatory properties.
We have not seensensitivity to endotoxin with the soluble glucan.
We have not seenthe inflammatory changesthat are so common with
zymosan.
Dr. Rodriguez,we did not objectify or quantitate the amount of contamination during operation in thesepatients. [t is of interest that the
glucan group had a total of si¡¡patients with colon injuries, whereasthe
placero group had only two patientswith colon injuries. AII colon injuries
were managedwith colostomy. Failure to quantitate contamination is a
weakpoint in the study, but on the surfaceit appearsthat the two groups
werecomparable.
The mechanicaI ventilator is a difficult paramenter to quantitate.
Overall it is very difficult to study patient groups that are 'similar' after
a traumatic insulto In the present study we individualized mechanical
ventilation managementin thesepatients and weanedthem offthe ventilators as rapidly as they could tolerate. It is interesting that the three
patientswho developedpneumoniascarneoffthe ventilator quite rapidly,
and we could not relatethe pneumonia to prolonged ventilation.
We did not look at PGE-2 levels in this particular study. We have
done many animal studies,suchas one using a hind limb crush model
in which glucanstimulation promptly increasedIL-I releasewhile actuaIly
decreasingPGE-2,so thereappearsto besome differentiaIin the response
of the macrophagein this setting.
1 would agreewith Dr. Rodriguez that theTNF levelsmight be time
related in the sensethat by measuringthem at inappropriate times or
not frequently enoughone might certainlymiss a very transient increase,
and thesestudiessimply haveto beextendedand aSsayed
in more detail.
Letme quote a Chineseproverb: 'A joumey of a thousandmiles begins
with a singlestep.' What we are seeingwith this studyand other clinical
studiesthat are evaluatingbiologic responsemodifiersare those first few
steps in that long joumey. I hope it will end with new benefits to the
trauma patient.