# Early Expression of FcγRI (CD64) on Monocytes  of Cardiac Surgical Patients and

Authors: Martina Kolackova; Manuela Kudlova; Pavel Kunes; Vladimir Lonsky; Jiri Mandak; Ctirad Andrys; Karolina Jankovicova; Jan Krejsek
Journal: Mediators of Inflammation
 (2008)

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## Abstract

Objective.
Activation of innate immunity cells is inseparably linked to cardiac surgical operation. The aim of this study was to assess the kinetics in the expression of receptor for Fc part of IgG, FcγRI (CD64), and scavenger receptor CD163 on peripheral blood cells of cardiac surgical patients and to examine the effect of cardiac bypass as a separable influence on the systemic acute inflammatory response.
Methods.
Forty patients, twenty in each group, were randomly assigned to CABG surgery performed either with  “on-pump” or without “off-pump” cardiopulmonary bypass. Standardized quantitative flow cytometry method was used to determine the expression of surface markers.
Results.
The density of CD64 molecule on monocytes reached maximum on the 1st postoperative day (P<.001) whereas the peak for CD64 molecule expression on granulocytes was postponed to the 3rd
postoperative day (P<.001).   The expression of CD163 scavenger molecule on monocytes reached maximum on the 1st postoperative day
(P<.001). The density of CD163 molecule on monocytes on the 1st postoperative day is
significantly higher in “on-pump” patients in comparison
with “off-pump” patients (P<.001).
Conclusion.
In cardiac surgical patients the expression of activation marker FcγR1 (CD64) on monocytes is increased earlier in comparison with granulocytes in
both “on-pump” and “off-pump” patients. The expression of scavenger
molecule CD163 on monocytes is significantly higher in “on-pump” patients.


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## Body

## 1. Introduction

Numerous  events, potentially
generating an inflammatory  response, are induced during cardiac surgery.
Amongst them, the combination of surgical injury, mechanical manipulation with
the heart, the contact of blood components with the artificial surfaces of the
cardiopulmonary bypass circuit, transient endotoxemia and ischemia-reperfusion
injury of the heart and lungs are relevant [1]. The inflammatory reaction is
the result of a complex interplay between numerous humoral factors and cell
substrate of inflammation. Amongst cells involved in this process special role
is devoted to innate immunity monocyte-macrophages and granulocytes. Whereas
monocyte-macrophage cells are the richest source of pluripotent proinflammatory
cytokines upon activation, activated granulocytes are recruited into tissues by
stepwise interaction between adhesion molecules on the surface of leukocytes
and their corresponding receptors expressed on the lumenal surface of inflamed
endothelium [2]. There is a substantial long lasting effort to identify
activated monocytes and neutrophils in blood of patients with systemic
inflammatory response induced by various stimuli either to identify patients at
the risk of development of overwhelming inflammatory response potentially
ultimating into multiple organ failure syndrome (MOFS) or to implicate the causative
agent of such inflammatory response, for example, bacterial infection [3].Activation
of myeloid cells by various physiological and experimental stimuli is
accompanied by multiple surface changes associated predominantly with
degranulation. Thus, activated blood myeloid cells typically upregulate surface
expression of chemotactic receptors, complement receptor type 3 (CD11b/CD18),
and downmodulate surface density of lipopolysacharide (LPS) receptor CD14, the
low affinity FcγRIII or CD16
receptor, adhesion receptors CD44 and CD62L [4].The Fcγ-receptor I, FcγRI, (CD64), is a high affinity receptor for
IgG1 and IgG3 subclasses of immunoglobulins. FcγRI is constitutively expressed with high
density on monocytes and macrophages, less so on eosinophils, but only to a
very low extent on resting neutrophils.Numerous
substances both exogenous
and endogenous origins
rapidly upregulate FcγRI expression
on the surface of neutrophils [5]. Microbial
cell wall components such as LPS,
endogenous complement split products, and cytokines,
such as IFNγ and TNFα, are some of the activators. The
expression of FcγRI is
determined by immunofluorescence and flow cytometry. The introduction of a new
diagnostic kit Leuko64 enables standardized and quantitative approach to the
determination of FcγRI expression
on immune cells.Hemoglobin scavenger
receptor CD163 is a group B cysteine-rich scavenger receptor expressed
exclusively by cells of monocyte-macrophage lineage. This glycoprotein is
characterized as a scavenger receptor for hemoglobin, mediating endocytosis of
hemoglobin-haptoglobin complexes. Previous studies have indirectly linked
CD163 scavenger receptor to anti-inflammatory phenomena [6].  High CD163 expression correlates with the Mo2
anti-inflammatory properties of monocytes and macrophages [7].The aim of this study was to follow
the changes in the expression of granulocyte activation markers FcγRI and anti-inflammatory CD163 scavenger
receptor in patients undergoing cardiac surgical operation either with the use
of cardiopulmonary bypass (“on-pump") or operated on the beating heart during
“off-pump" operation and in the postoperative period using quantitative flow
cytometric approach.


## 2. Patients

Forty patients (31 males,
mean age67.9±9 and 9 females, mean age 66.4±6.4, collective mean age 67.6±8.5 years) referred to first-time coronary
artery bypass grafting (CABG) were enrolled in this study. Patients underwent
either conventional myocardial revascularization with cardiopulmonary bypass
and cardioplegic arrest of the heart (“on-pump," n  = 20, 16 males, 4
females, mean age 69.4±7) or beating heart surgery (“off-pump,"  n =20,
15 males, 5 females, mean age 65.9±9.7).Patients in both groups were
comparable in age, preoperative left ventricular ejection fraction (median 0.65
in “on-pump," 0.65 in “off-pump" patients, resp.)   and the number of performed
coronary anastomoses (median 2.0 in “on-pump," 2.0 in “off-pump," resp.). The study protocol was approved by the
Ethics Committee of the University Hospital in Hradec Králové. All participants
were informed in detail about the purpose of the study both orally and in
writing. They were free to ask any questions. One person refused to participate
for reasons he would not specify. All active subjects have given written
informed consents.Cardiopulmonary
bypass, “off-pump" technique, and anesthesiological management have been
recently described in detail elsewhere [8].


## 3. Blood Sampling

Peripheral venous blood from an
antebrachial vein was withdrawn in the operating room and in the intensive care
unit. Samples were collected into heparinized tubes Vacutainer, Cat. no. 36884
manufactured by Becton Dickinson.In both “on-pump” and “off-pump” patients,
blood was withdrawn at the  following time points:(1)
introduction
to anaesthesia, which in both groups represented the baseline or reference
value for all parameters measured thereafter,(2)
after
termination of the operation,(3)
the
first postoperative day,(4)
the
third postoperative day,(5)
the
seventh postoperative day.
Additional samples were taken from “on-pump”
patients:(1a)
before cross-clamping of the aorta,(1b)
after aortic cross-clamp release,(1c)
after termination of CPB.


## 4. Materials and Methods

Leuko64
kit manufactured by Trillium Diagnostics, LLC, (www.trilliumdx.com, Brewer, Me, USA) was
used to determine the expressions
of CD64 and CD163 on leukocytes of blood samples. Leuko64 kit is composed of a
reagent cocktail of two monoclonal antibodies with specifities to CD64 (clones
22 and 32.2, both FITC conjugated) and monoclonal antibody to CD163 (clone
Mac2-148, phycoerythrin conjugated) and a fluorescence beads suspension used
for instrument calibration and standardization of leukocyte CD64 and CD163
expressions in human
blood. The assay was run according to the instruction for use provided by
manufacturer. Briefly, 50 microliters of blood and the Leuko64 monoclonal
cocktail reagent are incubated for 10 minutes, red cell lysis buffer is added
and incubated for additional
15 minutes, and 5 microliters of bead suspension are added prior to flow
cytometric analysis.  Results were measured by an FACSCalibur flow
cytometer (BD Biosciences, San Jose, Calif, USA) using CELLQuest software. The
listmode data were analyzed using Leuko64 software (Trillium Diag.). Results
are expressed as indexes of positivity for CD64 and CD163 expressions on
granulocyte and monocyte populations as provided by the Leuko64 software.


## 5. Statistical Analysis

We compared changes in the
intensity of expressions
of CD64 and CD163 in both groups of patients (“on-pump," “off-pump") separately.
Samples taken at the introduction to anesthesia were considered as reference or
baseline expressions
of CD64 and CD163. Differences between “off- and on-pump” patients were also
evaluated.Data were analyzed using two-way
ANOVA for repeated measures with Fisher test for multiple comparisons. To
exclude confounding effect of different age and sex presentation in both
groups, unpaired t-test and chi-square were performed. A probability (P)
value < .05 was considered significant. Statistical analysis was performed
with Statistica 5.5 software (Statsoft, Okla, USA).


## 6. Results

We
found substantial dynamics in the expressions of both CD64 and CD163 molecules on immune cells in
our cardiac surgical patients. These changes are expressed as changes in CD64
and CD163 indexes separately for monocytes and granulocytes. Preoperative
levels were taken as reference points. There were no significant changes in the
monocyte CD64 expression during cardiac operation in “on-pump” patients (data
not shown). The monocytes CD64 index was significantly increased in both “on-pump”
and “off-pump” patients in postoperative period from the first to the seventh
day (P  <  .01). Comparing monocyte CD64 index between “on-pump” and
“off-pump” patients, there were no significant differences (P  <  .587)
during operation and in the postoperative period
(Figure 1). The similar
patterns were found for monocyte CD163 index in our cardiac surgical patients
with following exceptions. Monocyte CD163 index, in contrast to the monocyte
CD64 index showed a significant difference as a function of the pump
status.  There was a statistically
significant increase in monocyte CD163 index in “on-pump” patients compared to “off-pump”
patients at the first postoperative day (P  <  .001)
(Figure 2).  The significant increase in monocyte CD163
index in both “on-pump” and “off-pump” patients compared to the baseline
expression was found only at the 1st and 3rd postoperative days (P  <  .01).Figure 1
CD64 molecule expression on monocytes in
“on-pump" and “off-pump" patients.Figure 2
CD163 molecule expression on monocytes in
“on-pump" and “off-pump" patients.The
granulocyte CD64 index was significantly increased both in “on-pump” and “off-pump”
patients at the 1st and 3rd postoperative days.
Statistically significant differences of granulocyte CD64 indexes
between “on-pump” and “off-pump” were not reached (P  <  .195)
(Figure 3).
The original and pathophysiologically very important observation in our results
is that the maximum of CD64 expression on monocytes (1st postoperative day)
precedes the maximum of CD64 expression on granulocytes (3rd postoperative
day).  The granulocyte CD64 expression
returned to baseline or normal levels by the 7th postoperative day.Figure 3
CD64 molecule expression on granulocytes in
“on-pump" and “off-pump" patients.


## 7. Discussion

A
long-term sustained effort is devoted to the better understanding of
inflammatory reaction which is inseparably linked to every cardiac surgical
operation. Numerous humoral and cell-mediated parameters of immune system have
been studied to identify markers describing the development of such
inflammatory reaction. The ultimate goal of such studies is to identify those
patients who are at substantial risk for development of overwhelming systemic
inflammation (SIRS), a condition of complex physiology that might severely
compromise the outcome of surgery or even lead to death. Many studies conducted
in the last few years have shown reduced inflammation in patients operated on
by the “off-pump” technique compared to “on-pump” surgery [9]. On the other
hand, any definitive proof in
favor of “off-pump” surgery in terms of reduced long-term mortality
compared to its “on-pump” counterpart has not been reported.  The very trauma of surgery seems to be more
relevant in initiating SIRS rather than cardiopulmonary bypass itself, the
latter adding a CPB specific fraction on top of unfavorable events [10].The
objective of this study was to compare the degree of activation of innate
immunity cells between “on-pump” and “off-pump” patients.  The novel diagnostic Leuko64 kit was used by
us to dissect such differences. This kit enables quantitative determination of
CD64/CD163 molecule expression on immune cells by flow cytometry.FcγR1 receptor (CD64) is constitutively expressed
on macrophages, monocytes, and eosinophils, but its expression is negligible on
resting neutrophils. Neutrophil CD64 molecule expression is one of many
activation-related surface receptors changes manifested during the normal
innate immunity response. Microbial cell wall components such as LPS,
complement split products, and cytokines (IFNγ, TNFα) are some of the activators [3].  In contrast to numerous other neutrophilic
activation surface antigens, such as CD11b/CD18, CD14, and CD16 with large
storage intracellular pools, CD64 has limited intracellular storage, but de
novo synthesis can be induced in the presence of proinflammatory conditions. In
comparison with former neutrophilic activation markers, CD64 expression on
neutrophils is thus much less affected by stimuli of degranulation to which
these cells are extensively exposed both during cardiac surgical operation and
during blood sample processing [5]. Thus CD64 molecule appears uniquely suited
as a surrogate marker of neutrophil activation or systemic acute inflammatory
response as its expression starts from less than 2000 sites per cell and
becomes upregulated in a graded fashion depending upon intensity of stimulation
by cytokines [4].Clinical
usefulness of CD64 determination has been proven in differential diagnosis of
sepsis of bacterial origin. Changes in the expression of CD64 on circulating
leukocytes in patients undergoing cardiothoracic surgery were also reported [11].
Some of these previous works are suffering from the lack of precise
quantitation since CD64 positivity was for example expressed as mean
fluorescence intensity [11, 12].  This
means of quantitation is valid on a single platform for day to day comparison,
but lacks standardization necessary for a routine clinical laboratory test,
whereas the Leuko64 kit approach does allow for interlaboratory comparisons.
Application of a new standardized diagnostic kit in our study overcomes above
mentioned limitations. Using calibration fluorescence beads and specialized
software, CD64/CD163 density is expressed as index positivity separately for
granulocytes and monocytes. As expected, CD64 positivity on monocytes is one
log higher in comparison with granulocytes. A significant increase in both
monocytes and granulocytes CD64 expressions has been found on the 1st and 3rd
postoperative days. No significant differences between “on-pump” and “off-pump”
patients have been recognized. Whereas CD64 expression on monocytes is nearly
identical for both “on-pump” and “off-pump” patients, there is a tendency for
higher granulocyte CD64 index on the 3rd postoperative day in “on-pump”
patients. This likely reflects the greater sensitivity to cytokine effects of
granulocytes compared to monocytes. Our results are in contrast to the work of
Stefanou et al. [13] who did not report any induction of
CD64 expression on
monocytes of cardiac surgical patients post CPB. However, only 10 patients were
enrolled to their study with some differences in CPB compared to our patients.
Furthermore, our attempt using Leuko64 kit ensures better standardisation of
method in comparison with previous work, where positivity was expressed as a
simple MFI.The neutrophils CD64 index is
designed so that normal inactivated cells yield value of <1.00 and blood samples from individuals with
documented sepsis or SIRS typically show values of >1.50. In our patients,
the perioperative and postoperative period was uneventfull with the only one
exception, the patient, who will be discussed below. It is our original
observation that the maximum of CD64 expression on monocytes of our cardiac
surgical patients has already been reached on the 1st postoperative day in
contrast to granulocytes in which the maximum of CD64 expression has been
postponed to the 3rd postoperative day. This observation implies that FcγRI expression on monocytes is upregulated by a
different cellular mechanism very early during operation by the exposition of
monocytes to various danger patterns which are raised during operation. The
upregulation of FcγRI on
granulocytes is a secondary event mediated by their exposition to
proinflammatory cytokines and mediators formed by monocytes-macrophages.The
FcγRI receptor
(CD64) on white blood cells integrate responses involving both the innate and
acquired immune systems and are very important for effective phagocytosis of
bacteria and immune complexes. A new, intrinsic role for this receptor has
recently been proposed by Devaraj et al. who have described the participation
of FcγR on
internalization of C-reactive protein by endothelial cells, with subsequent
release of chemoattractive proinflammatory IL-8, decrease of eNOS, and
increased ICAM-1 and VCAM-1 adhesion molecules [14]. In conclusion, FcγRI reveals proinflammatory activities in
general.Proinflammatory
pathways have to be tightly counterbalanced by numerous anti-inflammatory
processes, that is production of anti-inflammatory cytokines such as IL-10 [15],
to maintain protective physiological level of inflammatory response in cardiac
surgical patients. Another approach to calm down inflammatory reaction is
removing of pleiotropic proinflammatory species by the action of so-called
scavenger receptors which are differentially expressed on cells of both immune
and nonimmune origins [6]. Unique characteristics amongst these scavenger
receptors reveal hemoglobin
scavenger receptor (CD163). CD163 scavenger receptors represent a highly
efficient system to remove potentially toxic and proinflammatory
hemoglobin from the
circulation and local sites of inflammation [16]. Cardiac surgical patient are
extensively exposed to large amounts of free heme/hemeproteins due to
intravascular haemolysis and tissue damage. There is accumulating evidence that
an excess of free heme can cause cell damage and tissue injury. Heme catalyzes
the formation of reactive oxygen species (ROS), resulting in oxidative stress.
Because the low-molecular weight iron chelated heme is lipophilic, it can
easily intercalate in the membrane and impair lipid bilayers and organelles,
such as mitochondria and nuclei, and destabilize cytoskeleton [17]. Several
defense mechanisms against free heme-mediated oxidative stress and inflammation
exist. They consist of intra- (e.g., heme oxygenase-2 and heme oxygenase-3) and
extracellular (e.g., hemopexin, albumin) scavengers, antioxidative enzymes, and
heme oxygenase-1 during hemolysis.Intravascular
free hemoglobin is captured by plasma scavenger protein haptoglobin. It is
generally accepted that stable hemoglobin-haptoglobin complexes are subsequently delivered to
the reticuloendothelial system by CD163 receptor-mediated endocytosis [16]. Any
free vascular heme is bound to the plasma protein hemopexin or albumin, which transport
it to the liver for degradation in reticuloendothelial system. However, when
large amounts of free heme proteins or heme (locally) accumulate, like in a
blood clot or after vascular deposition, the scavengers get overwhelmed or are
unable to reach them. This enables heme to exert its damaging effects.
Therefore, the amount of free heme must be tightly controlled to maintain
homeostasis and avoid pathological conditions.The
monocyte CD163 index has been found to be between 5 000 and even 40 000 in our
study. There was a sharp increase of monocyte CD163 in the postoperative period
reaching statistically highly significant maximum (P  <  .001) on the 1st postoperative day for both
“on-pump” and “off-pump” patients followed by decrease to the baseline
preoperative levels. Furthermore, the patients’ monocyte CD163 index is
significantly higher (P  <  .001) in “on-pump” patient group on the 1st
postoperative day. Reports regarding monocyte CD163 expression in cardiac
surgical patients are very sparse. In a study by Goldstein et al. [18], a significant
increase in the monocyte CD163 expression on the 1st postoperative day was
found. This is in accord with results of our study. But there were several
limitations in a previous study. CD163 molecule was expressed as mean
fluorescence index (MFI) in comparison with our study in which standardized
quantitative approach using calibration fluorescence beads was exploited
yielded much more relevant data. The changes in the entire perioperative period
and during postoperative period up to the 7th postoperative day have been
followed by us in comparison with a previous study.The
link of CD163 hemoglobin scavenger receptor to anti-inflammatory phenomena in
cardiac surgical patients has been proven by Philippidis et al. [19]. In their
study, elevated expression of CD163 on circulating monocytes during the
resolution phase of the systemic inflammatory response to cardiopulmonary
bypass surgery was reported. Furthermore, binding of hemoglobin-haptoglobin
complexes to CD163  expressing monocytes elicited potent interleukin-10
secretion. It was reported by Goldstein et al. [18] that an increase in the monocyte
CD163 expression was 14 times higher in their “on-pump” patients who were
treated by bolus administration of metylprednisolone perioperatively in
comparison with untreated patients. Such therapeutical intervention was not
performed in any our patients. It seems unlikely that higher monocyte CD163
expression in our “on-pump” patients was elicited by metylprednisolone which is
a regular component of CPB fluids used at our department. More pronounced
proinflammatory stimuli raised during “on-pump” surgery are more relevant.
There will be the unique chance to dissect between the influence of
corticosteroids present in CPB fluid and other variables involved in “on-pump”
surgery. Currently, CPB protocol used at our department has been changed and
methylprednisolone has been ommited. CD163 is rapidly shed from the surface of
monocytes when activated by LPS or phorbol esters. In addition, also
cross-linking of FcγR triggers
shedding of CD163 [20]. This phenomenon
is very likely counterbalanced by the fact that such shedding is followed by
upregulation of this hemoglobin scavenger receptor, as was shown for metaloproteinases-mediated
CD163 shedding [21]. This is probably true for our cardiac surgical patients.
Very recently, it was proven by Weaver et al. [22] that CD163 shedding is also
induced via stimulation with TLR-4, TLR-2, and TLR-5.Initially
included in this study was a single patient, male, 73 years old who was
excluded from our study due to his death on the 2nd postoperative day. This
patient underwent uncomplicated cardiac surgery using cardiopulmonary bypass.
He developed acute diaphragmatic myocardial infarction two hours after
finishing surgery. Patient was reoperated and reanastomosis was performed. In
spite of this effort, cardiogenic and subsequent hemorrhagic shock was developed
ultimating to death next day in the morning. Body temperature was below36∘C so
that infection is unlikely.The
expression of both CD64 and CD163 molecules before surgery and up to the end of
surgery was comparable with other patients. Granulocyte CD64 index reached 1.72
value which is significant for the development of SIRS [23]. However, such
values were found in additional four patients without any impact on their
postoperative course. Remarkable is the fact that monocyte CD163 index in this
patient was the highest between our patients and was approximately two times
higher in comparison with all our patients investigated. It could be concluded
from this our anecdotal observation that the increase in CD163 monocyte
positivity could be the marker with predictive value of worse outcome in
cardiac surgical patients.In
conclusion, using standardized quantitative methods we revealed substantial
dynamics in the expression of the activation marker FcγRI and scavenger receptor CD163 in both
“on-pump” and “off-pump” cardiac surgical patients during operation and in the
postoperative period.  The maximum in the
expression of CD64 on monocytes in postoperative period precedes the maximum in
the expression of this molecule on granulocytes by two days.



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*Source: 235461-2008-02-19.xml*