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Differential Cellular Accumulation of Transforming Growth Factor–b1, –b2,
and –b3 in Brains of Patients Who Died with Cerebral Malaria
Martin H. Deininger,1 Peter G. Kremsner,2
Richard Meyermann,1 and Hermann J. Schluesener1
1
Institute of Brain Research and 2Department of Parasitology
(Institute of Tropical Medicine), Medical School, University
of Tübingen, Tübingen, Germany
In cerebral malaria (CM), pathologic cytokine expression patterns are thought to contribute
to disruption of the blood-brain barrier, inflammation, and astrocytic scar formation. Expression of transforming growth factor (TGF)–b1, –b2, and –b3 was analyzed in the brains
of 7 patients who died with CM and in 8 control patients. In the brains of patients with CM,
there were significantly (P = .0003 ) more TGF-b1–immunoreactive astrocytes adjacent to brain
vessels with deposition of malarial pigment, significantly (P = .0081 ) more TGF-b2–expressing
macrophages/microglial cells in glioses of ring hemorrhages and Dürck’s granulomas, and
significantly (P = .0022) more TGF-b3–expressing smooth-muscle cells and endothelial cells
of brain vessels with sequestration. It is concluded that focal accumulation of TGF-b1, -b2,
and -b3 provides evidence for their involvement in the reorganization process of the brain
parenchyma, immunologic dysfunction, and endothelial cell activation in patients with CM.
Plasmodium falciparum can cause the most severe form of
malaria infection in humans. Although most infected persons
suffer a mild febrile illness, some patients have clinical signs of
severe disease. Frequently observed clinical sequelae of severe
malaria infection include anemia, renal failure, scattered intravascular coagulation, shock, and respiratory distress. Cerebral
malaria (CM) is considered the most serious complication, with
a high mortality rate and up to 0.5 million deaths per year.
The clinical course of CM is characterized by reversible encephalopathy and loss of consciousness. The detailed pathophysiology of CM remains far from resolved; however, inflammatory cytokines have been suspected to contribute to CM
disease by modulating key parameters of brain parenchyma
homeostasis, such as endothelial cell integrity, activation of astrocytes, and immune response.
Among other factors, transforming growth factor (TGF)–b1,
–b2, and –b3 are thought to contribute to some of the phenomena observed in patients with malaria. In mice, TGF-bs
induce protective immune responses and slower parasite growth
early in infection and down-regulate pathogenic responses late
in systemic disease [1]. As a consequence, a reduction of peri-
Received 8 April 1999; revised 7 February 2000; electronically published
5 June 2000.
Informed consent was obtained from patients.
Grant support: Federal Ministry of Education, Science, Research and
Technology (Fö 01KS9602), Interdisciplinary Clinical Research Center, Tübingen; Fortüne program of the Faculty of Medicine, Tübingen.
Reprints or correspondence: Dr. Martin H. Deininger, Institute of Brain
Research, University of Tübingen, Medical School, Calwer Str. 3, D-72076
Tübingen, Germany ([email protected]).
The Journal of Infectious Diseases 2000; 181:2111–5
q 2000 by the Infectious Diseases Society of America. All rights reserved.
0022-1899/2000/18106-0042$02.00
pheral TGF-b concentrations during systemic malaria and CM
has been observed and may be associated with TGF-b tissue
accumulation [2]. Accordingly, a decreased expression of TGFb genes was observed in brains of mice susceptible to CM [3].
Only limited information is available about TGF-b function
in CM; however, there is convincing evidence that TGF-bs are
candidate cytokines involved in the formation of hallmark
pathophysiologic alterations in patients with CM. TGF-bs primarily modulate functional properties of cells that participate
in the immune response. In the brain, TGF-bs contribute to
global inflammation in experimental autoimmune encephalomyelitis [4], astrocytic scar formation [5], and endothelial cell
activation [6].
To evaluate a possible role of TGF-bs in CM, we analyzed
the expression of TGF-b1, -b2, and -b3 in the brains of 7
patients who died with CM and in 8 control patients (5 who
died without and 3 who died with neuropathologic alterations).
Patients and Methods
Patients. Specimens were collected from the brains of 7 Europeans who acquired P. falciparum malaria in Africa and died with
CM in primary care hospitals after returning to Germany. Computed tomography scans and lumbar punctures were done to ensure
the clinical diagnosis of CM and to exclude other infectious diseases
of the brain. P. falciparum was detected in the peripheral venous
blood smears of all patients. No differences were observed in the
48-h red blood cell–stage cycle among the patients. All patients
were in the late part of the trophozoite and schizont stage. The
clinical condition of the patients was monitored using the Glasgow
coma scale. Prominent intravascular sequestration with parasitized
erythrocytes could be observed readily in all examined tissue specimens. The 8 controls included 5 patients who died without neuro-
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Deininger et al.
pathologic alterations, 1 who died with fatal bacterial meningitis,
1 who died with active demyelinating multiple sclerosis, and 1 who
died with Alzheimer’s disease. Samples were obtained from affected
regions of each brain that were characterized either by granuloma
formation or intravascular sequestration.
Immunohistochemistry. Brain samples were obtained from subjects shortly after death, fixed in buffered formalin, and embedded
in paraffin. Sections (5 mm) were deparaffinized and rehydrated.
For antigen retrieval, the sections were immersed in 0.01 M citrate
buffer and irradiated during 5 cycles of 5 min in a 750-W microwave
oven. Endogenous peroxidase was blocked with 1% H2O2 in methanol, and then the slices were incubated with nonspecific porcine
serum. Monospecific polyclonal rabbit antibodies directed against
human TGF-b1 and -b2 (Santa Cruz, Santa Cruz, CA) and monoclonal mouse anti–human TGF-b3 antibody (Oncogene Research
Products, Cambridge, MA) were diluted in 1% bovine serum albumin (BSA) and Tris balanced salt solution (TBS; pH 7.5; containing 0.025 M Tris and 0.15 M NaCl) at dilutions of 1 : 50 (TGFb1), 1 : 200 (TGF-b2), and 1 : 10 (TGF-b3). The diluted antibodies
were added to the sliced brain samples and incubated for 1 h at
room temperature. Secondary antibody (biotinylated anti–rabbit
IgG or biotinylated anti–mouse IgG; Dakopatts, Glostrup, Denmark) was diluted 1 : 400 in BSA-TBS and applied to the slices
for 30 min. Streptavidin-biotin–horseradish peroxidase complex
(Dakopatts), diluted 1 : 400 in BSA-TBS, subsequently was applied
for 30 min. Labeled antigen was visualized with standard diaminobenzidine techniques (Sigma, St. Louis) and was consecutively
counterstained with hematoxylin.
Double-labeling experiments. In double-labeling experiments,
we first labeled a cell type–specific antigen, using the avidin-biotin–
complex (ABC) procedure in combination with alkaline-phosphatase conjugates. In brief, slices were deparaffinized, irradiated in a
microwave oven for antigen retrieval, and incubated with nonspecific porcine serum as described above. Then the differentiating
antibodies directed against glial fibrillary acidic protein (GFAP;
Boehringer Mannheim, Mannheim, Germany), leukocyte common
antigens HLA-DR, -DP, and -DQ (major histocompatibility complex class II), CD68 (macrophages), CD3 (T cells), CD20 (B cells),
von-Willebrand factor (vWF; endothelial cells), sm-actin (smoothmuscle cells), and S-100b (astrocytes) (all from Dakopatts) were
added to the slices at a dilution of 1 : 100 in TBS-BSA. Visualization was achieved by adding biotinylated rabbit anti–mouse IgG
or biotinylated swine anti–rabbit IgG, both diluted 1 : 400 in BSATBS for 30 min, and alkaline phosphatase–conjugated ABC complex diluted 1 : 400 in BSA-TBS for 30 min. For developing, we
used Fast Blue BB salt chromogen-substrate solution (Sigma-Aldrich, Deisenhofen, Germany), which yielded a blue reaction product. Between double-labeling experiments, slices were irradiated in
a microwave oven for 20 min in citrate buffer. Alkaline phosphatase
was inhibited completely as described elsewhere [7]. Following these
steps, TGF-bs were immunolabeled as described above.
Analyses and controls. Ten regions of granuloma and intravascular sequestration were evaluated at 3500 magnification for
each antibody that was applied. In control brains, corresponding
regions in the white and gray matter were counted. Samples with
no positive cells were assigned score 0, samples with <2% labeled
cells were assigned score 1, samples with 2%–10% labeled cells were
assigned score 2, samples with 10%–20% labeled cells were assigned
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score 3, samples with 20%–50% labeled cells were assigned score
4, and samples with 150% labeled cells were assigned score 5. Mean
labeling scores (MLSs) were calculated and compared by use of
the 2-tailed Mann-Whitney rank sum test.
Single-labeling immunohistochemistry controls included incubation of the tissue slices with nonimmune TBS-BSA and blocking
experiments. Specificity of antibodies was confirmed by Western
blot analysis and immunoprecipitation, as confirmed by the
manufacturers.
Results
TGF-b1, -b2, and -b3 in neuropathologically unaltered control
brains. In neuropathologically unaltered brain sections (table
1), TGF-b1 primarily was expressed in resting macrophages/
microglial cells and in scattered astrocytes. Double-labeling experiments revealed the coexpression of HLA-DR, -DP, and
-DQ in macrophages/microglial cells or the coexpression of
GFAP in astrocytes. TGF-b2 expression was detected in few
resting macrophages/microglial cells and occasionally in neurons scattered throughout the cortical gray matter. TGF-b3
expression again was found predominantly in single macrophages/microglial cells and in few neurons. Only occasionally,
endothelial cells in 2 patients showed weak TGF-b3
immunoreactivity.
TGF-b1, -b2, and -b3 in brains of patients with CM. In
brains of patients who died with CM (table 1), prominent TGFb1 expression was detected in astrocytes that form the bloodbrain barrier around cerebral capillaries characterized by deposition of malarial pigment and sequestration (figure 1A).
Double-labeling experiments revealed the coexpression of S100b (astrocytes) but not of HLA-DR, -DP, and -DQ or CD68
(macrophages/microglial cells) in TGF-b1–expressing cells. Statistical analysis using the 2-tailed Mann-Whitney rank sum test
revealed significantly (P = .0003 ) more TGF-b1–expressing astrocytes in brain samples from patients with CM (MLS, 4.71;
SE, 0.18) than in neuropathologically unaltered brain samples
(MLS, 1.13; SE, 0.12).
TGF-b2 was found in macrophages/microglial cells that accumulated in Dürck’s granulomas and in glioses of ring hemorrhages (figure 1B). TGF-b2–expressing cells were characterized by the coexpression of CD68 and HLA-DR, -DP, and -DQ
but not of S-100b. Perivascular TGF-b2–expressing cells were
double labeled only with CD68. No colocalization of HLADR, -DP, or -DQ was observed in these cells. Statistical analysis
revealed significantly (P = .0081 ) more TGF-b2–expressing
macrophages/microglial cells in brain samples from patients
with CM (MLS, 3.25; SE, 0.48) than in neuropathologically
unaltered brain samples (MLS, 1.12; SE, 0.13). Accumulation
of TGF-b2–expressing cells was found occasionally in areas of
perivascular cellular infiltration. Furthermore, few TGF-b1–
and -b2–expressing neurons and macrophages/microglial cells
were scattered in the examined brain parenchymas.
TGF-b3 expression was prominent in endothelial cells and
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TGF-bs in Cerebral Malaria
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Table 1. Immunohistochemical transforming growth factor (TGF)–b1, –b2, and –b3 labeling
scores of patients who died with cerebral malaria and of control patients who died without
neuropathologic alterations.
Group, cause of death/histology
Control
Myocardial infarction
Cardiac failure
Pulmonary embolism
Cardiac arrest
Myocardial infarction
Fatal menigitis
Multiple sclerosis
Alzheimer’s disease
Cerebral malaria
Parasitized erythrocytes in cerebral
microvasculature, small Dürck’s
granulomas adjacent to vessels,
petechial hemorrhages
Small Dürck’s granulomas adjacent
to the vasculature, no hemorrhages,
parasitized erythrocytes within
microvasculature
Parasitized erythrocytes in cerebral
microvasculature, several ring
hemorrhages
Parasitized erythrocytes, sludge
phenomenon, petechial and
ring hemorrhages, Dürck’s
granuloma
Brain edema, parasitized erythrocytes
in microvasculature, no Dürck’s
granulomas, no ring hemorrhages
Parasitized erythrocytes in
vasculature, no hemorrhages,
no Dürck’s granulomas
Parasitized erythrocytes in
vasculature, no hemorrhages,
no Dürck’s granulomas
TGF-b1 in
astrocytes
TGF-b2 in macrophages/
microglial cells
TGF-b3 in
endothelial cells
1
2
1
1
1
1
1
1
1
1
2
1
2
1
1
0
1
0
0
0
1
1
2
0
5
4
2
4
2
3
5
4
2
5
3
3
4
1
3
5
2
1
5
1
3
NOTE. Labeling score was determined as follows: 0, no staining; 1, up to 2% labeled cells; 2, up to 10%
labeled cells; 3, up to 20% stained cells; 4, up to 50% labeled cells; and 5, 150% labeled cells.
smooth-muscle cells of the brain vasculature of patients with
CM that was characterized by deposition of malarial pigment
and sequestration (figure 1C). TGF-b3 expression in endothelial
cells was confirmed by colocalization of vWF. TGF-b3–
expressing cells of the vascular wall were double labeled for
sm-actin. Colocalization of the T cell marker CD3 and the B
cell marker CD20 was not observed in TGF-b1–, TGF-b2–,
and TGF-b3–expressing cells. Statistical analysis revealed significantly (P = .0022) more TGF-b3–expressing endothelial
cells in brain samples from patients with CM (MLS, 2.43; SE,
0.29) than in neuropathologically unaltered brain samples
(MLS, 0.62; SE, 0.26).
TGF-b1, -b2, and -b3 in brains of patients with multiple sclerosis, Alzheimer’s disease, or bacterial meningitis. In the brain
specimen of the patient who died with active demyelinating
multiple sclerosis, TGF-b1 expression was detected predominantly in astrocytes of demyelinated plaque areas. TGF-b2 immunoreactivity was observed occasionally in macrophages/microglial cells. Weak TGF-b3 immunoreactivity was detected in
endothelial cells.
Brain slices of the patient who died with Alzheimer’s disease
were characterized by focal accumulation of TGF-b1–
expressing astrocytes. TGF-b2 immunoreactivity, in contrast,
was observed only in scattered macrophages/microglial cells.
TGF-b3 immunoreactivity was observed in astrocytes but not
in endothelial cells.
In the patient who died with bacterial meningitis, TGF-b1
immunoreactivity was observed predominantly in a small cortical margin immediately adjacent to areas of cellular infiltration. TGF-b2 and -b3 immunoreactivity was detected predominantly in macrophages/microglial cells in areas of cellular
infiltration. Few astrocytes expressed TGF-b3.
Discussion
Compared with control brains without neuropathologic alterations, the brains of patients who died with CM had cell
type–specific accumulation of TGF-b1, -b2, and -b3. In normal
brains, we observed TGF-b1 expression in scattered macrophages/microglial cells and astrocytes, and TGF-b2 and -b3
Figure 1. Brain sections of patients who died with cerebral malaria. A, Transforming growth factor (TGF)–b1 expression (brown color) found
in astrocytes that form a blood-brain barrier around cerebral capillaries characterized by deposition of malarial pigment and sequestration. B,
TGF-b2 (brown color) expression found in macrophages/microglial cells in Dürck’s granulomas and in glioses of ring hemorrhages. C, TGF-b3
expression (brown color) found in endothelial and smooth-muscle cells in capillaries with deposition of malarial pigment and sequestration. All
slices were counterstained with hematoxylin. Bars, 25 mm.
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TGF-bs in Cerebral Malaria
expression in few macrophages/microglial cells and neurons. In
the brains of patients with active demyelinating multiple sclerosis, Alzheimer’s disease, or bacterial meningitis, we observed
cell type–specific accumulation of TGF-b1, -b2, and -b3 immunoreactivity, as partly described elsewhere [8, 9].
The gross effects of TGF-b1 in the brain are aimed at the
reconstruction of neural tissues after their disruption during
inflammation and degeneration and the maintenance of neuronal viability [10]. In detail, TGF-b1 induces apoptosis of microglial cells [11], controls central nervous system (CNS) inflammation by regulating cytokine-induced vascular cellular
adhesion molecule–1 expression [12], and promotes angiogenesis by eliciting vascular endothelial growth factor expression
[13]. Therefore, our findings provide further evidence for the
involvement of TGF-b1 in the reorganization process of the
brain parenchyma in patients with CM. Accumulation of TGFb2–expressing macrophages/microglia cells in Dürck’s granulomas and in glioses of ring hemorrhages elicits new functional
insights into the detailed pathophysiology of these lesions.
TGF-b2 is considered to interfere with key functions of the
immune system. In the brain, TGF-b2 mediates immunosuppression, inhibits leukocyte transmigration across the bloodbrain barrier in CNS inflammation, and promotes intracerebral
macrophage proliferation. [14]. Accumulation of TGF-b2–
expressing macrophages/microglial cells in granulomas and
glioses of ring hemorrhages therefore indicates complex interactions with the immunologic dysfunction observed in these
patients.
TGF-b3 expression in endothelial and smooth-muscle cells
is considered an important modulator of cellular activation and
transformation, and reorganization of complex vascular structures has been thought to involve, at least in part, TGF-b3.
Only limited data are available on the involvement of TGF-b3
in brain pathologies; however, expression of TGF-b3 was identified in pathologically altered smooth-muscle cells of atherosclerotic vessels and in fibrotic vasculature of hepatic human
allografts and therefore has been associated with thrombosis,
atherosclerosis, and endothelial cell activation [15]. Immunolocalization of TGF-b3 in the brain vasculature characterized
by deposition of malarial pigment and sequestration in patients
who died with CM therefore suggests its involvement in sequestration and thrombosis.
In contrast to the brains of patients who died with multiple
sclerosis, Alzheimer’s disease, or bacterial meningitis, the brains
of patients who died with CM had specific TGF-b1, -b2, and
-b3 expression patterns that provide evidence for the differential
involvement of TGF-b isoforms in the reorganization process
of the brain parenchyma, immunologic dysfunction, and endothelial cell activation.
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Acknowledgment
We thank Thai Dung Nguyen for expert technical assistance.
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