1. Art and Diseño

Effects on Leukocytes After Injection of Tumor Necrosis Factor
Into Healthy Humans
By Tom van der Poll, Sander J.H. van Deventer, C. Erik Hack, Gert J. Wolbink, Lucien A. Aarden,
Harry R. Buller, and Jan W. ten Cate
Tumor necrosis factor (TNF) has been implicated as a proximal mediator of the septic syndrome. To evaluate the possible role of TNF in leukocyte activation in septicemia, we
performed a cross-over saline-controlled study in six healthy
men who were intravenously injected with recombinant
human TNF (50 pg/m*), and analyzed changes in circulating
white blood cells and parameters for neutrophil and monocyte activation. TNF elicited a very rapid neutropenia, reaching a nadir after 15 minutes, followed by a neutrophilia.
Lymphocytes showed a sustained decrease, whereas monocytes declined transiently. TNF injection was also associated
with neutrophil activation, as reflected by a mean fivefold
increase in the plasma concentrations of elastase-q-antitryp-
sin complexes and a mean sevenfold increase in plasma
lactoferrin levels. Serum neopterin, a marker of monocyte
activation, was significantly increased 24 hours after the
administration of TNF. These changes occurred in the absence of detectable complement activation, as indicated by
unchanged C3a-desarg plasma values. Serum interleukin-6
showed a nearly 40-fold increase after TNF injection, whereas
interleukin-1 remained undetectable throughout. We conclude that the systemic release of TNF, triggered early after
invasive infection, may be involved in the alterations in
circulating leukocyte numbers and in the activation of leukocytes, during the development of the septic syndrome.
o 1992 by The American Society of Hematology.
I
TNF in these inflammatory reactions. For this purpose we
NVASIVE BACTERIAL infection may give rise to the
clinical syndrome of septicemia characterized by fever,
hypotension, vascular leakage, and ultimately multiple
organ failure.’ Several physiologically operative host defense mechanisms have been implicated in the development of this syndrome, including leukocytes, which may
cause tissue injury by releasing toxic substances from their
granules and reactive oxygen metabolites.’ The extent of
neutrophil activation in septicemia has been shown to have
a prognostic ~ignificance.’.~
In the past years it has become clear that invading
bacteria trigger the production of small polypeptides, collectively termed cytokines, mainly by mononuclear and endothelial cells. Among these cytokines, tumor necrosis factor
(TNF), interleukin-1 (IL-1), and IL-6 play an important
role in the pathophysiology of septicemia? Several lines of
evidence indicate that TNF is a critical factor in the
initiation of the septic syndrome. Systemic TNF release is
observed early after infusion of live bacteria in animals6and
endotoxin in humans? and high serum levels of TNF are
found in patients with sepsis.8Administration of recombinant TNF induces the septic syndrome in animals,” while
monoclonal antibodies (MoAbs) directed against TNF
prevent mortality in lethal sepsis models, concurrently
attenuating the elaboration of IL-lp and IL-6 into the
circulation.”’
Recent in vitro studies have shown interactions between
the cytokine network and leukocytes, ie, cytokines are
capable of activating a variety of leukocyte functions,”
granulocytes can produce cytokine~,’~~’’
and cytokines can
enhance the expression of complement receptors on leukoc y t e ~This
. ~ ~ suggests that organ damage in septicemia may
result from an interplay between these components of the
immune system. We recently studied the kinetics of leukocyte activation after an intravenous injection of endotoxin
into healthy
Endotoxin induced a biphasic
change in leukocyte counts involving initial neutropenia
and subsequent neutrophilia, associated with degranulation
of neutrophils as reflected by increases in the plasma
concentrations of elastase-ai-antitrypsin complexes and
la~toferrin.’~’~
Interestingly, these responses occurred in the
absence of detectable complement activation.’ In the present
Blood, Vol79, No 3 (February I ) , 1992: pp 693-698
investigation, we aimed to assess the role of circulating
studied six healthy men in a saline-controlled cross-over
fashion after a bolus intravenous injection of recombinant
human TNF.
MATERIALS AND METHODS
The study was approved by the institutional research and ethics
committees of the Academic Medical Center, University of Amsterdam, and written informed consent was obtained from all volunteers. All subjects were admitted to the Metabolic Research Ward
and confined to bed during the 12-hour study period.
Study design. The present study was performed simultaneously
with investigations on the coagulative and endocrine effects of
TNF, results of which have been published previ~usly.’~.’~
The
outcomes of clinical parameters, ie, increases in body temperature,
occurrence of chills, and the absence of hemodynamic changes
were also reported in
Six healthy male volunteers (age 27 to 33 years) participated in
the study. Medical history, physical examination, and routine
laboratory investigation were completely normal in all subjects.
They did not use medication and had no febrile disease in the
month before the study.
Each study period started at 7:30 AM. The volunteers fasted
overnight until the end of the study. Each subject was studied twice
with an interval of at least 3 weeks. On one occasion a bolus
From the Department of lnternal Medicine and the Center for
Hemostasis, Thrombosis,Atherosclerosis and Inflammation Research,
Academic Medical Center, University of Amsterdam; and the Central
Laboratoiy of the Netherlands Red Cross Blood Transfusion Service
and the Laboratoiy for Experimental and Clinical Immunology,
University of Amsterdam, Amsterdam, The Netherlands.
Submitted April 1,1991; accepted September 23, 1991.
S.J.H.V.D.and H.R.B. are fellows of the Royal Netherlands Academy of Arts and Sciences.
Address reprint requests to T. van der Poll, MD, Department of
Internal Medicine, Academic Medical Center, F4-222, Meibergdreef 9,
1105 A 2 Amsterdam, The Netherlands.
The publication costs of this article were defiayed in part by page
charge payment. This article must therefore be hereby marked
“advertisement” in accordance with 18 U.S.C.section 1734 solely to
indicate this fact.
0 1992 by The American Society of Hematology.
0006-497119217903-0014$3.00/0
693
VAN DER POLL ET AL
694
intravenous injection of recombinant human TNF of 50 pg/m2
dissolved in 10 mL of isotonic saline was administered; on the other
occasion an equivalent volume of isotonic saline was administered.
The order in which recombinant TNF and isotonic saline was given
was determined by balanced assignment.
Venous blood samples were obtained by separate venipunctures,
with the use of 19-gauge butterfly needles, directly before the
injection of recombinant TNF or isotonic saline and 15,30, and 45
minutes and 1, 2, 3, 4, 5, 6, 8, 10, and 12 hours thereafter.
Additional samples were obtained 24 hours postinjection. Blood
for the measurement of serum TNF concentrations was obtained
immediately before the injection of recombinant TNF or saline and
5,10, 15,30,60, 120,180, and 240 minutes thereafter.
Recombinant human TNF was kindly provided by Boehringer
Ingelheim (Ingelheim am Rhein, Germany). It was more than 99%
pure as determined by sodium dodecyl sulphate-polyacrylamide gel
electrophoresis analysis and contained less than 10 ng of endotoxin
per milligram of protein as tested by the limulus amoebocyte lysate
assay.
Assays. Leukocyte counts were determined in blood anticoagulated with K,-EDTA with the use of a flow cytometer (Technicon
H1 system; Technicon Instruments, Tarrytown, NY). Leukocyte
count differentials were determined both by flow cytometry and
hand counting of peripheral blood smears, which showed identical
results. Blood for the determination of elastase-a,-antitrypsin
complexes, lactoferrin, and C3a-desarg was collected in siliconized
Vacutainer tubes (Becton Dickinson, Plymouth, UK) to which
EDTA (10 mmol/L) and Polybrene (Aldrich, Milwaukee, WI)
(0.05%, wt/vol) were added to prevent any in vitro complex
formation. Blood samples were centrifuged at 4°C for 20 minutes at
1,600g. The plasma concentrations of elastase-a,-antitrypsin complexes and lactoferrin were measured with a radioimmunoassay
(RIA) that has been described in detail? Briefly, sepharose beads,
to which polyclonal antibodies against human elastase or an MoAb
against lactoferrin were coupled, were incubated with the samples
to be tested. Elastase-a,-antitrypsin or lactoferrin bound to the
beads was quantitated by incubation with '"I-MoAb against complexed a,-antitrypsin (RIA for elastase-a,-antitrypsin) or polyclonal I-antilactoferrin (RIA for lactoferrin). The plasma levels
of elastase-a,-antitrypsin complexes and lactoferrin are expressed
as nanograms per milliliter using preformed complexes and purified lactoferrin as standards, respectively. Complement activation
was assessed by measuring the plasma concentrations of C3adesarg by an RIA as reported previously.18To prevent interference
with native C3 in the assay, samples were incubated with polyethylene glycol before being tested. The results are expressed as
nanomoles per liter.
Serum for the determination of neopterin, TNF, IL-6, and IL-1
was prepared by centrifugation of clotted blood for 20 minutes at
1,600g (room temperature). The serum concentrations of neopterin were measured with an RIA (IMMUtest Neopterin; Henning,
Berlin, Germany) and expressed as nanomoles per liter. The serum
levels of TNF were determined by IRMA (Medgenix, Fleurus,
Belgium) as described previously.16 Polypropylene tubes were
coated with a combination of MoAbs to recombinant TNF that
recognize distinct epitopes of TNF. The tubes were incubated
overnight with a mixture of the sample to be tested and anti-TNF
antibody labeled with '"I. After decantation, the bound fraction
was counted in a gamma counter, and the level of TNF was
expressed in picograms per milliliter in relation to a standard
binding curve for recombinant TNF. The serum concentrations of
IL-6 were measured using the B9 assay as described previously."
Briefly, serum was heated for 30 minutes at 56°C and a titration of
each serum was added to 5,000 B9 cells and compared with a
standard IL-6 preparation. Serum IL-6 levels are expressed as units
per milliliter, where 1 U/mL is the concentration that leads to
half-maximal proliferation. One unit equals about 1pg of IL-6. The
identity of IL-6 in positive sera was confirmed with a neutralizing
antiserum to recombinant IL-6. The limit of detection of the B9
assay is 7 U/mL. Samples with values below the limit of detection
were assigned a value of 7 U/mL. The serum concentrations of
IL-1 were measured using the D10 assay as reported previously.*"
D10 cells were incubated with serum in the presence of IL-2 (50
U/mL). Under these conditions 1 pg of IL-1 per milliliter causes
half-maximal proliferation. Standard curves were generated by
incubation with recombinant IL-la. The limit of detection of the
D10 assay is 30 pg/mL.
Statistical analysis. Values are given as means f SEM. Differences in results between the TNF and saline experiments were
tested by analysis of variance and Newman-Keul's test for multiple
comparison, as indicated. A P value <.05 was considered to
represent a significant difference.
RESULTS
Leukocyte counts. Baseline leukocyte counts were similar and within normal limits in both study periods (Fig 1).
TNF injection was associated with a biphasic change in
total leukocyte counts, which largely reflected the change in
the number of circulating neutrophils (both P < .0001 by
analysis of variance). Marked initial decreases in total
leukocytes and neutrophils were observed, reaching a nadir
after 15 minutes. Total leukocytes dropped from 5.2 f 0.4
x 10y/Lat baseline to 1.2 2 0.1 x 10y/L;neutrophils from
2.6 f 0.3 X 10y/L to 0.2 & 0.04 X 10y/L. Early blood
sampling in three subjects showed that the decrease in
circulating neutrophils was already apparent as early as 5
minutes after TNF injection. The initial neutropenia was
followed by a neutrophilia becoming significant after 1
hour. From this timepoint and onward band cells appeared
in the circulation, with a maximum of 18% to 26% of
circulating white blood cells after 2 to 4 hours. Total
leukocyte and neutrophil counts peaked after 6 hours
(12.9 f 1.3 x 10y/L and 11.9 ? 1.3 x 10y/L, respectively)
and were still significantly elevated after 12 hours. Total
leukocyte and neutrophil counts had returned to control
values in samples obtained after 24 hours (data not shown).
TNF administration also elicited a rapid and sustained
lymphopenia (P < .0001 by analysis of variance), which
reached a nadir after 5 hours (from 2.0 f 0.2 X 10y/Lat
baseline to 0.3 ? 0.1 x 109/L).Lymphocytes remained decreased until the end of the 12-hour study period; 24 hours
after TNF or saline injection lymphocyte counts were
similar (data not shown). TNF induced a transient monopenia lasting 2 hours postinjection (P < .005 by analysis of
variance). Minimal monocyte counts were observed after 15
minutes, when monocytes had almost completely disappeared from the peripheral blood (from 0.3 f 0.1 X 10y/L
at baseline to 0.02 f 0.01 x 109/L).
Neutrophil activation. Activation of neutrophils was assessed by measurements of the plasma concentrations of
elastase-a,-antitrypsin complexes and lactoferrin. Preinjection values were similar in both study periods (Fig 2). TNF
elicited sharp increases in the plasma levels of elastaseqantitrypsin complexes and lactoferrin, both becoming significant after 30 minutes and peaking after 3 hours. Elastase-
TNF AND LEUKOCYTES
695
At baseline, neopterin levels were not different in both
study periods (6.0 & 1.3 nmol/L before TNF; 5.3 f 0.4
nmol/L before saline), and they remained unchanged until
8 hours after the initial injections. Twelve hours after TNF
injection a modest nonsignificant increase in serum neopterin was found compared with the control period (7.8 f 1.4
nmol/L v 5.8 f 1.2 nmol/L), which became significant 24
hours postinjection (8.7 & 0.8 nmol/L v 4.5 f 0.8 nmol/L).
Complement activation. Activation of the complement
system was monitored by measuring the plasma concentrations of C3a-desarg. As compared with saline, TNF did not
affect C3a-desarg levels throughout the entire observation
period (data not shown).
Cytokines. The serum levels of TNF have been reported
previously.’6 Briefly, TNF was not detectable in serum
obtained at baseline or during the saline control period (Fig
3). After injection of recombinant TNF, the highest serum
level was measured after 5 minutes (4,261 f 785 pg/mL).
Thereafter, serum TNF concentrations decreased rapidly.
Leukocytes (xlOO/l)
128-
40-
Neutrophils (XIOO/l)
12-
84-
-
0.
Elastase -a,antltrypsin (ng/ml)
Lymphocytes (xlOa/I)
‘1
-
* * * * *
*
l5OI
0
Monocytes (XIOg/l)
0
0.6
J*I
I
1
Lactoferrin (ng/ml)
*T
0
2
4
6
8
1
0
1
*T
2
Tlme (hrs)
Fig 1. Mean (kSEM) leukocyte counts after intravenous bolus
injections of recombinant human TNF (50 pg/mZ; solid circles) or an
equivalent volume of isotonic saline (open circles). Asterisks indicate
statistical significance for the comparison of TNF with saline (P < .05
by Newman-Keul‘s test).
a,-antitrypsin increased from 43 f 6 ng/mL at baseline to
223 f 28 ng/mL ( P < .0001 by analysis of variance);
lactoferrin from 212 f 41 ng/mL to 1526 f 227 ng/mL
(P < .0001 by analysis of variance). From 3 hours and
onward gradual decreases in elastase-a,-antitrypsin and
lactoferrin were observed, whereby the former remained
elevated until the end of our 12-hour observation period.
Monocyte activation. The serum concentrations of neopterin were determined as a measure of monocyte activation.
,
0
2
4
6
a
I
.
.
12
Time (hrs)
Fig 2. Mean (+SEM) plasma concentrations of elastase-a,antitrypsin complexes and lactoferrin after intravenous bolus injections of recombinant human TNF (50 pglm’; solid circles) or an
equivalent volume of isotonic saline (open circles). Asterisks indicate
statistical significance for the comparison of TNF with saline (P < .05
by Newman-Keul’s test).
VAN DER POLL ET AL
696
- 300
- 200
- 100
‘I
0
2
4
8
.
.
-0
,
8
12
Tbne (Ius)
Fig 3. Mean (+SEM) serum concentrations of IL-6 after intravenous bolus injection of recombinant human TNF (50 pg/mz; solid
circles); IL-6 remained below the limit of detection (7 U/mL) in the
saline control period. Asterisks indicatestatistical significancefor the
comparison of TNF with saline (P < .05 by Newman-Keul‘s test). In
addition, mean (+SEM) serum levels of TNF are shown (solid triangles) after injection of recombinant TNF. TNF was not detectable
during the control period.
Serum IL-6 concentrations were below the limit of
detection (7 U/mL) in the saline control period. TNF
induced an increase of serum IL-6 concentrations that
became significant after 30 minutes (Fig 3). Serum IL-6
peaked after 2 hours (265 4 56 U/mL; P < .OW1 by
analysis of variance).
IL-1 activity was not detectable in serum during either
study period (data not shown).
DISCUSSION
TNF is a pleiotropic protein that occupies a pivotal early
role in the pathogenesis of the septic syndrome. In the
course of septicemia, initiation of the cytokine network is
followed by activation of other mediator systems, which is
considered to importantly contribute to the development of
the septic syndrome. Among these mediator systems, leukocytes are of particular interest. The objective of the present
study was to evaluate the effects of circulating TNF on
white blood cells. It was shown that intravenous injection of
TNF induced marked changes in peripheral blood leukocyte counts, and activation of neutrophils as reflected by up
to sevenfold increases in the plasma concentrations of
elastase-a,-antitrypsin complexes and lactoferrin. An increase in neopterin serum levels after 24 hours indicated
monocyte activation.
TNF elicited an early decrease in circulating neutrophils
that was already apparent after 5 minutes and reached a
nadir after 15 minutes. Thereafter, neutrophil counts rapidly increased and a neutrophilia with immature forms
developed. The initial neutropenia was most likely caused
by adhesion of neutrophils to the vascular endothelium. In
vitro TNF almost instantly enhances the adherence of
neutrophils to cultured endothelial cells by increasing the
expression of the CD 11/18 complex on the surface of
neutrophils.” The neutrophilia from 1 hour and onward
was probably due mainly to recruitment of marrow neutrophils, because in rats TNF-induced neutrophilia is associated with decreased neutrophil counts in the bone marrow.22Additionally, in cancer patients, labeled autologous
leukocytes reinfused 15 minutes before TNF infusion rapidly disappeared from the circulation and did not return
when leukocytosis developed.23 TNF injection also provoked a sustained lymphopenia, which may have been
caused by enhanced lymphocyte adhesion to vascular endothelium secondary to expression of intercellular adhesion
molecule 1 (ICAM-1) and vascular adhesion molecule 1
(VCAM-1) on the surface of endothelial cells by a direct
action of TNF.24,25
Although IL-1 remained undetectable in
the circulation after TNF injection, confirming previous
findings in cancer patients infused with TNF in higher
doses,26 cell-bound IL-1 may have been involved as a
secondary mediator in the TNF-induced changes in circulating white blood cells as it shares many effects with TNF on
the interaction between leukocytes and e n d o t h e l i ~ m . ~ ~ ~ ~
IL-6 does not affect the adhesive capacity of endothelial
cells,x but has been shown to induce neutrophilia and a
mild lymphopenia in rats in vivo.*’ Since, in the present
study, serum IL-6 showed a nearly 40-fold increase after
TNF injection, this cytokine may have contributed to
altered neutrophil and lymphocyte counts observed several
hours after the administration of TNF.
Activation of neutrophils likely plays a pivotal part in the
development of organ damage in septicemia. The capability
of neutrophils to disrupt the normal architecture of tissues
is derived from the combined cooperative action of released
chlorinated oxidants and proteinases2 Elastase is a very
potent proteinase, degrading almost all components of the
extracellular matrix, as well as a variety of plasma proteins.28 Moreover, elastase is an important mediator of
endothelial cell injury, resulting in vascular leakage.2yThe
host’s primary defense against uncontrolled action of elastase is the proteinase inhibitor a,-antitrypsin, which rapidly
and irreversibly binds to elastase, forming elastase-a,antitrypsin complexes. Lactoferrin is a glycoprotein from
neutrophil specific granules. Several proinflammatory functions have been postulated for lactoferrin, including antimicrobicidal activity and regulation of neutrophil adhesiveness. Neutrophils secrete elastase and lactoferrin on
stimulation with various agonists in vitro. As neutrophils
are the predominant source of circulating
and
the exclusive source of circulating la~toferrin,~’
the plasma
concentrations of these proteins are commonly used as a
measure of neutrophil activation in vivo. In patients with
sepsis, elevated plasma levels of elastase-a,-antitrpsin
complexes and lactoferrin have been consistently f o ~ n d . 3 , ~
TNF induced marked increases in the plasma concentrations of both neutrophil markers, reaching significance
after 30 minutes and peaking after 3 hours. TNF may have
provoked this neutrophil degranulation directly,””’ at later
stages possibly in concert with IL-6.33In addition, granulocyte-macrophage colony-stimulating factor (GM-CSF), an
important stimulator of the proliferation and maturation of
leukocytes, may have been involved because TNF induces
the production of this protein by several cell types in vitro?4
697
TNF AND LEUKOCYTES
We did not measure GM-CSF in serum because, to our
knowledge, release of GM-CSF to the circulation has never
been found in infectious or other diseases.
Intravenous endotoxin has also been shown to induce
increases in the plasma levels of elastaseq-antitrypsin and
1a~toferrin.l~’~
However, in these experiments the increase
in elastase-a,-antitrypsin occurred only after 2 to 3
whereas lactoferrin started to increase after 90 minutes.15
The differential time courses of the increases in neutrophil
markers after endotoxin and TNF injection indicate that
degranulation of neutrophils in endotoxemia may proceed
via endotoxin-induced TNF. These results, taken together
with previous studies reporting that TNF stimulates respiratory burst activity of neutrophils in vitro36 and primes
neutrophils for hypochlorous acid production in vivo?
implicate this cytokine as an important factor in the
activation of neutrophils in septicemia.
Neopterin is exclusively released by activated monocytes
and macrophages. Patients with septic shock have very high
levels of neopterin in the circulation, which is interpreted to
reflect monocyte activation?8 Our study establishes that
TNF may play a role in this process. TNF induced an
increase in neopterin serum concentrations, becoming significant after 24 hours, which contrasted with the rapid
activation of neutrophils. It is likely that TNF stimulated
neopterin release indirectly, as it does not affect the
production of neopterin by monocytes in
Severe septicemia is commonly associated with activation
of the complement ~ystem.~’
Intravenous injection of TNF
did not elicit complement activation, as indicated by unchanged plasma levels of C3a-desarg. This finding confirms
our previous observation after injection of endotoxin into
healthy subjects, that activation of granulocytes can occur in
the absence of complement activati~n.’~’~
It is unlikely that
our inability to detect complement activation was due to
inappropriate sensitivity of the assay for C3a-desarg. Using
the same assay we could readily detect activation of the
complement system in several disease states, including
sepsis3’ and after treatment with recombinant IL-2 in
cancer patients:’ However, these patients were severely ill
and showed signs of shock and multiple organ failure, which
contrasts with the relatively mild clinical response to
intravenous endotoxin and TNF in healthy subjects. Conceivably, complement activation only occurs in more severe
clinical conditions. It remains to be established whether
repeated and/or prolonged exposure to higher amounts of
TNF does stimulate the complement cascade.
In conclusion, this study shows that a single intravenous
injection of TNF induces marked alterations in the peripheral blood counts and functional properties of leukocytes.
These data substantiate the proximal role of TNF in the
initiation of leukocyte activation in septicemia.
ACKNOWLEDGMENT
We are indebted to Dr Auguste Sturk and the other members of
the staff of the coagulation laboratory for their excellent technical
support; to Dr Frans J. Hoek for the determinations of the serum
concentrations of TNF; to Dr Fanny Berends for analyzing
peripheral blood smears; to Gerdie Wentink and Lida Stuiver for
preparing the illustrations; and to Marieke Kat for secretarial
assistance.
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