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From the Unité de Recherche "Immunopathologie Humaine," INSERM U430, and Claude Bernard Association, Hôpital Broussais, Paris, France
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Abstract |
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 Top Abstract IntroductionMethodsResultsDiscussionReferences |
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,
interleukin-1ß, and interleukin-6). IVIg inhibited
proliferation of endothelial cells in a time-dependent manner. This
effect was dependent on both Fc and F(ab')2 fragments of
the immunoglobulin molecule and was fully reversible. Tumor necrosis
factor- and interleukin-1ß also inhibited thymidine
incorporation, but to a lesser degree. IVIg had no effect on
basal levels of mRNA coding for the adhesion molecules,
chemokines, and proinflammatory cytokines. IVIg fully
down-regulated the expression induced by tumor necrosis factor- or
interleukin-1ß of mRNA coding for these molecules. Thus,
blockade of cellular proliferation and of cytokine-induced expression
of adhesion molecules, chemokines, and cytokines may
explain the therapeutic effect of IVIg in vascular and inflammatory
disorders.
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Introduction |
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TopAbstractIntroductionMethodsResultsDiscussionReferences |
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and -ß (TNF- and TNF-ß) play a
key role and can be induced in vitro by numerous agents such
as proinflammatory cytokines IL-1ß and TNF-,5-7
modified low-density lipoproteins,8
and bacterial
lipopolysaccharide.9
Intravenous immunoglobulin (IVIg) is therapeutic immunoglobulin (Ig)
prepared from pools of plasma of several thousand healthy blood donors.
In addition to its use as substitutive therapy for primary and
secondary antibody deficiencies, IVIg exhibits immunomodulatory effects
in diseases mediated by autoantibodies and in diseases believed to be
primarily mediated by autoaggressive T cells in humans and in
experimental animals.10,11
IVIg has been used effectively
in the treatment of autoimmune cytopenias,12-17
the acute
Guillain-Barré syndrome,18,19
myasthenia
gravis,20
and anti-factor VIII autoimmune
disease.21
Patients suffering from systemic inflammatory
conditions such as dermatomyositis,22
and, particularly,
Kawasaki syndrome greatly benefit from IVIg treatment (for review, see
23 and 24
). IVIg has also been used in the treatment of
anti-neutrophil cytoplasmic antigen-associated systemic
vasculitis.25,26
The mechanisms of action of IVIg are, as
yet, poorly understood, although several mutually nonexclusive
hypotheses have been proposed.11
These include the blockade
of Fc
receptors on phagocytic cells,27
interference with
activated complement,28,29
modulation of production and
release of cytokines and their inhibitors,30,31
modulation
of T- and B-lymphocyte functions,32-34
suppression of
autoantibody production, and selection of immune
repertoires.35,36
However, little is known on the direct
interaction between IVIg and ECs of the vascular bed. The present study
was undertaken to address the potential role of IVIg on EC function by
following both EC proliferation and EC expression of key adhesion
molecules, chemokines, and cytokines. We have shown that IVIg inhibited
EC proliferation in a dose- and time-dependent manner and
down-regulated the expression of adhesion molecule mRNA (ICAM-1 and
VCAM-1), chemokine mRNA (MCP-1, M-CSF, and GM-CSF), and proinflammatory
cytokine mRNA (TNF-, IL-1ß, and IL-6) induced by TNF- or
IL-1ß. These results may explain, at least in part, the
therapeutic effect of IVIg in vascular and inflammatory disorders.
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Methods |
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TopAbstractIntroductionMethodsResultsDiscussionReferences |
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IVIg (Sandoglobulin; Sandoz, Basel, Switzerland), was a kind gift from the Central Laboratory of the Swiss Red Cross Blood Transfusion Service (Bern, Switzerland). Two other preparations of IVIg were Gammagard (N. V. Baxter S. A., Lessines, Belgium) and Endobulin (Immuno AG, Vienna, Austria). F(ab')2 fragments were prepared from IgG by digestion with 2% (w/w) pepsin (Sigma Chemical Co., St Louis, MO) in acetate buffer, pH 4.1, for 18 hours at 37°C, followed by chromatography on protein A-Sepharose. F(ab')2 fragments were free of IgG and Fc fragments as assessed by sodium dodecyl sulfate-polyacrylamid gel electrophoresis and enzyme-linked immunosorbent assay. Concentrations of purified IgG and F(ab')2 fragments were determined spectrophotometrically at 280 nm. Fc fragments were a gift from Dr. M. C. Bonnet (Pasteur Merieux, Lyon, France). Human albumin was obtained from the Laboratoire Français de Fractionnement et de Biotechnologies, L. F .B. (Les Ulis, France). No endotoxin contamination was detected in IVIg preparations using the limulus amebocyte assay.31
Cell Culture
Umbilical cords were collected from healthy newborns after normal pregnancy and delivery (Notre-Dame de Bon Secours Hospital, Paris, France). Human umbilical vein ECs (HUVECs) were obtained after a 4-minute treatment of the umbilical vein with 0.15% collagenase I (300 U/mg, Sigma) in phosphate-buffered saline (PBS; 13.8 mmol/L NaCl, 0.5 mmol/L Na2HPO4, 4.1 mmol/L KCl, and 0.2 mmol/L KH2PO4) supplemented with 11.1 mmol/L glucose. The cells were cultured in 0.2% gelatin-coated (Sigma) 75-cm2 tissue culture flasks (Costar, Cambridge, MA) in medium M199 (Life Technologies, Inc., Grand Island, NY) containing Earle's salts, L-glutamine, and 25 mmol/L HEPES and supplemented with 20% fetal calf serum (FCS; Dutscher, Brumath, France), 100 U/ml penicillin, 100 µg/ml streptomycin, and 0.25 µg/ml fungizone (Life Technologies). At confluency, primary cultured cells were harvested after 6 minutes of trypsinization using 33% trypsin (Biological Industries, Kibbutz Beit Haemek, Israel) in PBS and plated again in three 75-cm2 tissue culture flasks from one flask. The ECs were characterized by their typical cobblestone morphology and by the presence of factor VIII antigen. For experiments, cells were used at passages two and three.
Growth-Inhibition Studies
After trypsinization, cells were seeded in 96-well plates (Costar)
coated with 0.2% gelatin, at a concentration of 1.5 x
104
cells/well and grown for 3 to 4 days in M199 containing
20% FCS. To synchronize cells in a G0/G1
stage, FCS concentration was reduced to 1% for 24 hours. At the end of
the depletion period, cells were replaced in M199/20% FCS and
stimulated for various periods of time in the presence of 0.5 µCi
[3H]thymidine/well (Amersham Life Science, Little
Chalfont, United Kingdom), with the following agents: 1) IVIg [10, 20,
30, and 40 mg/ml (0.10, 0.13, 0.20, and 0.26 mmol/L, respectively)]
for 6, 18, 24, 30, or 48 hours; 2) human albumin (40 mg/ml) for 48
hours; 3) F(ab')2 (0.20 and 0.26 mmol/L) and Fc (0.20 and
0.26 mmol/L) fragments of IVIg for 24 hours; and 4) purified human
recombinant TNF- (0.5 or 50 ng/ml; Alexis Corp., San Diego, CA),
purified human recombinant IL-1ß (0.5 or 50 ng/ml; Sigma), TNF-
(50 ng/ml) plus IVIg (40 mg/ml), and IL-1ß (50 ng/ml) plus IVIg (40
mg/ml) for 24 hours. At the end of incubation times, radioactivity was
measured by liquid scintillation counter (1450 MicroBeta Plus; Wallac,
Turku, Finland). The choice of IVIg concentrations in these in
vitro studies was based on the average concentrations of
therapeutic IgG found in the plasma of patients treated with IVIg.
Cell Viability
For determination of cell number, cells were seeded at a concentration of 5 x 103 cells/well and processed as above. After a 24- or 48-hour incubation period with 0, 20, 30, or 40 mg/ml IVIg, cells were harvested by a 6-minute trypsinization. Both live and dead cells were counted in a Malassez chamber using trypan blue. Cell viability was also assessed by FACScan after incubation in presence of IVIg (20, 30, or 40 mg/ml) or human albumin (40 mg/ml) for 24 or 48 hours. At the end of the incubation time, nonadherent cells were harvested, and adherent cells were trypsinized, washed twice with PBS, and suspended with the nonadherent cells in PBS at a final concentration of 1.5 x 105 cells/ml. Propidium iodide (10 µl; 500 mg/ml) (Sigma) was added to the 0.5 ml cell suspension, and the uptake of the dye by dead cells was immediately followed by fluorescence analysis using a FACScan (Becton Dickinson, San Jose, CA).
Reversibility Assay
Cells were seeded and starved as described. For reversibility assays, they were incubated in M199/20% FCS with or without IVIg as follows: 1) IVIg (20, 30, or 40 mg/ml) for 24 or 48 hours, and 2) IVIg (20, 30, or 40 mg/ml) for 24 or 48 hours followed by withdrawal of IVIg for the next 24 or 48 hours. In all cases, 0.5 µCi [3H]thymidine/well was added to the cultures for the last 24 hours of incubation period, and the radioactivity was measured.
Reverse Transcription and Polymerase Chain Reaction (PCR) Analysis
ECs were plated in 25-cm2
flasks in M199/20% FCS for
3 to 4 days to confluency. The medium was then replaced with control
medium (M199/20% FCS) and the agonists as follows: IVIg (1, 10, or 40
mg/ml), IL-1ß (0.5 or 50 ng/ml), TNF- (0.5 or 50 ng/ml), IL-1ß
(50 ng/ml) plus IVIg (40 mg/ml), and TNF- (50 ng/ml) plus IVIg (40
mg/ml). After 4 hours, mRNAs were extracted with 1 ml of TRIzol (Life
Technologies) for 5 to 10 x 106
cells, according to
the technique provided by the manufacturer. They were reverse
transcribed into cDNA with oligo(dT) and Moloney murine leukemia virus
reverse transcriptase (Life Technologies). For each experiment, the
volume of sample of cDNA was adjusted in such a way as to yield
identical levels of glyceraldehyde-3-phosphate dehydrogenase (GAPDH).
The reverse transcription products were amplified with primers listed
in Table 1
. All sequences were found in
the GenBank database (National Center for Biotechnology Information,
National Institutes of Health, Bethesda, MD). All PCRs were carried out
in 25 µl of a mixture containing 10 mmol/L deoxynucleotide
triphosphate, 1x PCR buffer (10 mmol/L Tris-HCl, pH 8.3, 50 mmol/L
KCl, 40% dimethyl sulfoxide, 0.001% gelatin, MgCl2 (see
concentrations in Table 1
), and 2.5 U AmpliTaq polymerase (Perkin-Elmer
Corp., Norwalk, CT)). Each sample was incubated in a DNA thermal cycler
(Perkin-Elmer Corp.) at 52°C to 58°C for 30 to 40 cycles, depending
on the primer (Table 1)
. The PCR fragments were analyzed by
electrophoresis on 2% agarose gels and visualized by ethidium bromide
staining (Eurobio, Les Ulis, France). Polaroid photographs of ethidium
bromide-stained gels were digitized into 512 x 512-pixel
gray-scale images. The amount of nucleic acid, determined by
densitometric analysis of the dots, was proportional to the logarithm
of the optic density. Analysis was performed using the public domain
NIH Image 1.51 program. The intensities of the cDNA bands for each
protein were normalized to the GAPDH band intensities. All experiments
represent at least three umbilical cords in culture. For each
cell extract, PCR was run five times.
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Proliferative response experiments were performed on at least three umbilical cords, with each value being measured in triplicate. Results are expressed as mean ± standard error of the mean. Statistical analysis was carried out using two-way analysis of variance with time of incubation or concentration of agents and treatment as factors. Statistical significance was achieved if P was < 0.05. In cases of interaction between the factors, one-factor analysis of variance was used at one level of the other factor. Data were analyzed using the Statview 4.0 software (Abacus Concepts, Inc., Berkeley, CA).
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Results |
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TopAbstractIntroductionMethodsResultsDiscussionReferences |
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, IVIg
(Sandoglobulin) significantly inhibited EC proliferation, as assessed
by [3H]thymidine incorporation. The inhibition was time
dependent and highly effective during the first 6 hours of treatment.
Between 6 and 30 hours, 10 mg/ml IVIg had no effect any longer, whereas
other concentrations induced, at a lower rate, an inhibition of
thymidine incorporation. After 30 hours of incubation, despite the
presence of IVIg, inhibition partly disappeared, except at the highest
IVIg concentration. A similar pattern of inhibition of the
proliferative response of HUVECs was also observed with two other
sources of IVIg, namely, Gammagard and Endobulin (Figure 2)
. The inhibitory effect of all of the
Ig preparations studied was dose dependent (Figures 1 and 2)
. Gammagard
and Endobulin were more potent inhibitors than Sandoglobulin (Figure 2)
.
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. When cells were incubated in the
presence of 20 or 30 mg/ml IVIg, but not with 40 mg/ml IVIg, the live
cell number was always higher after 48 hours than after 24 hours of
incubation, a feature that reflects the partial removal of
inhibition of thymidine incorporation seen after 30 hours. The
reduction of cell number is also partly due to the death of the cells
that increased with IVIg concentration, which, however, did not exceed
8% at the end of the treatment of cells with IVIg (duration of
treatment effect, P = 0.004; concentration effect,
P = 0.001; n = 3). When cell viability
was assessed by FACScan analysis (Figure 4)
, the number of dead cells as
determined by propidium iodide uptake did not exceed 6%, whatever the
time of incubation and/or the concentration of agonists (duration of
treatment effect, P = 0.45; group effect,
P = 0.38; n = 3). Therefore, the
significant decrease in the number of live cells and the small number
of dead cells during the entire period of treatment with IVIg indicated
that the inhibition of proliferation of cells is associated with a
blockade of cell division rather than only being due to cell death. To
check whether this inhibitory effect of IVIg was not simply due to the
presence of high amounts of nonspecific protein, 40 mg/ml of human
albumin instead of IVIg was added to the culture medium for 48 hours.
Thymidine incorporation remained similar to control under such
conditions (data not shown).
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illustrates the effect of
removal of IVIg from culture medium on EC proliferation. After a 24- or
a 48-hour incubation period of cells with IVIg,
[3H]thymidine incorporation was monitored at 0, 24, or 48
hours after IVIg withdrawal. When cells were incubated for 24 hours
with 20 and 30 mg/ml IVIg (Figure 5A)
, the inhibition of cell
proliferation disappeared and the uptake of thymidine was fully
restored 24 hours after IVIg had been withdrawn. However, when IVIg was
used at a 40 mg/ml concentration, a 24-hour washout was unable to
overcome the inhibition, which was lifted if recovery was pursued for
another 24-hour period. When IVIg was used for 48 hours at the
concentration of 20 or 30 but not 40 mg/ml (Figure 5B)
, thymidine
incorporation was partially restored at the time of IVIg withdrawal
(t = 0), as already seen in Figure 1
. The
inhibition level was then 1.5- to 2-fold less pronounced than just
after a 24-hour stimulation. Twenty four hours after IVIg removal, the
uptake of thymidine was fully recovered, whatever the concentrations of
IVIg.
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. Figure 6
shows the effect of TNF- and IL-1ß
on cell proliferation. After 24-hour incubation, TNF- significantly
reduced [3H]thymidine incorporation in a dose-dependent
manner, whereas IL-1ß was fully effective at a dose as low as 0.5
ng/ml. However, for both cytokines, the maximum inhibitory effect was
twofold less pronounced than the effect induced by 40 mg/ml IVIg
(44.7 ± 1.8, 43.2 ± 3.9, and 80.6 ± 2% inhibition
induced by TNF-, IL-1ß, and IVIg, respectively; n
= 3). HUVECs were then either coincubated with a mixture of TNF- or
IL-1ß and IVIg, prepared just before addition onto the cells (Figure 6
, lane A), or were pretreated for 15 minutes with TNF- or IL-1ß
alone, before the addition of IVIg into the culture medium (Figure 6
,
lane B). Under both conditions, IVIg plus cytokines led to a more
efficient inhibition of cell proliferation than IVIg alone (group
effect, P = 0.0012; n = 3), but no
difference was seen depending on the way in which IVIg was added
to the cytokines.
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. In concentrations ranging from 1 to 40
mg/ml, IVIg had no significant effect on the synthesis of mRNA encoding
ICAM-1 and VCAM-1. Both IL-1ß (0.5 and 50 ng/ml) and TNF- (0.5 and
50 ng/ml) induced a synthesis of ICAM-1 and VCAM-1 mRNA in a
dose-dependent manner. Again, IL-1ß was efficient at doses as low as
0.5 ng/ml. As compared with control, IL-1ß (50 ng/ml) and TNF- (50
ng/ml) increased twofold the expression of VCAM-1 and ICAM-1 mRNA.
Although IVIg had no substantial effect on the basal level of
expression of ICAM-1 and VCAM-1, IVIg (40 mg/ml) significantly
down-regulated to control values the expression of VCAM-1 and ICAM-1
mRNA induced by IL-1ß or TNF-.
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, IL-6, and IL-1ß). At rest, the expression of mRNA coding
for the chemokines (Figure 8)
and
cytokines (Figure 9)
of ECs was minimal.
IVIg had little or no effect on the basal level of chemokines and
proinflammatory cytokines. As shown in Figures 7 and 8
, IL-1ß and
TNF- significantly increased the levels of all chemokines and
cytokines studied. IVIg at 40 mg/ml down-regulated the expression
of the chemokines MCP-1, M-CSF, and GM-CSF and of the cytokines
TNF-, IL-6, and IL-1ß, induced by IL-1ß and TNF-.
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Discussion |
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TopAbstractIntroductionMethodsResultsDiscussionReferences |
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- or IL-1ß-induced expression of mRNA
encoding major adhesion molecules, chemokines, and proinflammatory
cytokines, which are significantly implicated in the leukocyte
recruitment observed in several inflammatory diseases.4 The observed inhibitory effect of IVIg on EC proliferation, as assessed by [3H]thymidine incorporation and cell enumeration, was not merely due to a high concentration of protein, because human albumin at 40 mg/ml had no effect on the thymidine uptake by the cells. The inhibitory effect of IVIg was also reversible, in a dose- and time-dependent manner. At 48 hours of culture of ECs in the presence of IVIg however, there was an abolition of the inhibitory effect of IVIg, and the cell number progressively increased. Although the mechanism underlying this escape of cells from the antiproliferative action of IVIg after 48 hours is not clear, the phenomenon reflects the clinical picture after therapy with IVIg: a transient decrease of leukocyte count followed by a recovery in the count has been observed in volunteers infused with IVIg.37 The drop in the number of live cells and the small increase in the number of dead cells clearly indicated that the inhibition of cell proliferation induced by IVIg is associated with an arrest of the cell cycle at the G0/G1 phase, rather than only being due to a mortality of the cells. Therefore, the changes in osmolarity provoked by the presence of sugar as a stabilizing agent and the acidic pH used to prevent the precipitation of commercial IVIg preparations had little effect on HUVEC viability. The mechanisms involved in the cell cycle arrest and mortality of cells remain unknown. We dissected the role of the variable region of Ig (F(ab')2 fragments) and the constant Fc portion of Ig in the antiproliferative effect of IVIg. We found that both F(ab')2 and Fc were able to inhibit significantly, in a similar level, the proliferation of HUVECs. Although the underlying mechanisms involved in the analogous effect observed with both F(ab')2 and Fc portions of Ig are not clear as yet, a receptor-mediated mechanism cannot be ruled out. In vivo, it is most likely that both F(ab')2 and Fc portions of Igs are involved in the immunomodulatory functions.38
The endothelium plays a central role in the immunopathology of several
vascular disorders in many inflammatory conditions such as Kawasaki
disease, Wegener's granulomatosis, or vasculitides in which use
of IVIg has been shown to be beneficial (for reviews, see 23, 39,
and 40). It may act either as a target for injury or by encouraging the
development of lesions because of its anatomical position and
physiological function. Bound anti-EC antibodies in Kawasaki disease
have the potential to mediate EC injury and lysis via either complement
or by more subtle changes in EC functions. It is well established that
the onset of inflammation provokes the expression of adhesion molecules
on ECs.4,7,41,42
As the basal level of expression of
adhesion molecules, chemokines, and cytokines by HUVECs under the
experimental conditions used in this study is low (Figures 7 through 9)
, we have induced the expression of the adhesion molecules ICAM-1 and
VCAM-1; the chemokines MCP-1, M-CSF, and GM-CSF; and the
proinflammatory cytokines TNF-, IL-1ß, and IL-6 by two
proinflammatory cytokines, IL-1ß and TNF-, to evaluate the effect
of IVIg on the expression of these molecules. The expression of TNF-
by HUVECs has been a matter of debate.43-48
Our
observation suggests that TNF- induces an autocrine up-regulation of
the mRNA expression of TNF-. The role of such an autocrine
production of TNF- on EC function is, however, not known. IVIg
significantly down-regulated the cytokine-induced expression of all
these molecules involved in an inflammatory process, although IVIg
alone, in concentrations ranging from 1 to 40 mg/ml, had no effect. One
of the reasons for this blocking effect may be the anti-cytokine nature
of IVIg, as IVIg contains antibodies directed against
cytokines.49,50
However, we did not observe any difference
in the proliferative responses of HUVECs, either when IVIg was mixed
with TNF- or IL-1ß before addition to the cells or when it was
added after a preincubation of cells with the cytokines for 15 minutes.
We therefore believe that the inhibitory effect of IVIg on the
cytokine-induced activation of HUVECs may not be exclusively due to
neutralization of TNF- or IL-1ß by antibodies directed against
these cytokines. Another possibility is that IVIg contains soluble
receptors that "soak up" the TNF- or IL-1ß stimulators that
abrogate the stimulatory effects of these cytokines. Further, it is
also possible that in IVIg, anti-idiotypic antibodies bearing internal
images of molecules that mimic such receptors may exist. The molecular
mechanisms involved in the modulation of the function of ECs warrant
further investigation.
Over the last decade, IVIg has been used in the treatment of several
posttransplantation complications including cytomegalovirus
(CMV)-associated diseases, including transplant arteriosclerosis,
obliterative bronchiolitis, CMV-induced myocarditis, CMV-associated
graft versus host disease, CMV-retinitis, and CMV-hepatitis
(acute and chronic).51-55
An immune-mediated vascular
disease is associated with acute and chronic allograft rejection, in
which T-lymphocyte activation and the release of interferon- by
activated T cells in turn induce the expression of class II molecules
on vascular ECs. Underlying mechanisms point out a relationship between
CMV infection and inducible expression of human leukocyte antigens on
allograft vascular ECs and on specific lymphocyte subpopulations that
infiltrate the graft.56
Such activation of ECs results in
an enhanced cellular adhesion and in lymphocyte-mediated tissue damage.
Our results suggest that some of the anti-inflammatory effects observed
in patients treated with IVIg are related to a decreased ability of ECs
to proliferate and to a down-regulation of the expression of molecules
involved in the onset and progression of inflammation. Although the
relevance of these findings in in vivo situation needs
further investigation, a possible beneficial effect of IVIg lies in the
control of EC activation in the inflammatory conditions, because
generation of microvessels is a salient feature of neoplasia and
inflammation.57,58
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Acknowledgements |
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Footnotes |
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Supported in part by grant 97043 from Rhône-Poulenc Rorer (Paris, France) and the Central Laboratory of the Swiss Red Cross (Bern, Switzerland). CX was supported by 24-month fellowship no. 3Q4/044 from the French Foreign Ministry and the French Embassy in Beijing, China.
This work was presented in abstract form at the European Renal Association, European Dialysis and Transplant Association Annual Congress, Geneva, Switzerland, September 21–24, 1997, and at the American Society of Nephrology Meeting in San Antonio, TX, November 2–5, 1997.
CX's present address is Department of Histology and Embryology, Shanghai Second Medical University, Shanghai, China. NL's present address is Laboratory of Experimental Surgery, Department of Clinical Surgery, Federal University of Santa Catarina, Florianopolis, Brazil.
Accepted for publication July 17, 1998.
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References |
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induce distinct patterns of endothelial activation and associated leukocyte accumulation in skin of Papio anubis. Am J Pathol 1989, 135:121-133[Abstract]
globulin. N Engl J Med 1982, 306:1254-1258[Abstract]
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