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From the Department of Pathology, University of Geneva, Geneva, Switzerland
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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TNF production is induced by an immune response, elicited by the presence of the parasite in the blood.13 Indeed, depletion of CD4 T lymphocyte prevents the acute mortality of PbA infection in mice, apparently by decreasing TNF production.1,13 TNF might be responsible for some of the manifestations of SM, such as the hypoglycemia and the sequestration of cells in the microcirculation. TNF is known to increase the expression of the adhesion molecules CD54 (ICAM-1) and CD106 (VCAM) on endothelia,14-16 which might contribute to increasing the adhesion of leukocytes and other cells and thereby disturbing the microcirculation in the brain and other organs. This pathogenic hypothesis is supported by the increased expression of CD54 and CD106 in the brain microcirculation during SM and the delayed mortality seen in mice treated with anti-CD11a mAb (LFA-1, a ß2 integrin determinant)6,17 or in CD54-deficient mice.18
CD40 is a cell receptor belonging to the TNF receptor superfamily that can modulate cell proliferation, differentiation, and death.19 Studies based on mice genetically deficient in CD40 or its ligand CD40L (CD154) as well as the use of anti-CD40L mAb have demonstrated that this system plays an important role in both humoral and cell-mediated immunity.20,21 Presence of CD40 has been reported on B lymphocytes, platelets, mast cells, endothelial cells, and dendritic cells, whereas the source of CD40L includes T lymphocytes, macrophages, and platelets.21,22 Response to infectious agents; rejection of allograft; autoimmune diseases, such as encephalitis, arthritis, atherosclerosis and pulmonary fibrosis, are attenuated in mice with a perturbation of the CD40-CD40L signaling.21 Understanding of the role of CD40-CD40L in cell-mediated immunity is presently incomplete because CD40-CD40L seems to be critical for the resistance to some intracellular parasites such as Leishmania,23 but not others such as mycobacteria.24 CD40-CD40L signaling has been reported to induce the expression of adhesion molecules CD54, CD62E, and CD106 on endothelial cells from the umbilical vein,25 that might be relevant to the pathogenesis of SM, as discussed above.
In this report, we explored the role of CD40-CD40L in the course of PbA-induced SM. Mortality was completely abrogated in CD40-/-, CD40L-/-, as well as in mice treated with the anti-CD40L mAb, indicating an essential role of this system in the pathogenesis of SM.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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CD40L-/- and CD54-/- mice,6 isolated on the C57BL/6 background, were obtained from the Jackson Laboratory (Bar Harbor, ME). CD40-/- were obtained from R. Geha, Boston, MA.26 Mice were bred in our animal facilities as well as the C57BL/6J (B6) also obtained from the Jackson Laboratory, which were used as wild-type (+/+) controls.
PbA and Treatment
PbA has been passed in rodents1 and mice were infected by an intravenous injection of 5 x 104 parasitized red blood cells (pRBCs).
Anti-CD40L mAb was derived from the hybridoma MR1 of hamster origin.27 mAbs or nonimmune hamster IgG, as a control, were purified by protein A-Sepharose. Mice were injected with the anti-CD40L mAb or nonimmune hamster IgG (250 µg IgG, ip) on day 6 after infection.
Blood
Blood (0.015 ml) was isolated from the retro-orbital plexus of ethrane-anesthetized mice using heparinized capillaries and diluted in ethylenediaminetetraacetic acid (1% final), in accord with the Swiss national guidelines. Blood elements were counted in a cell counter (Casy 1, Schärfe system, D-72760 Reutlingen, Fluka, 9470-CH). Parasitemia was counted on blood smears stained with May-Grunwald-Giemsa.
Assessment of Vascular Leak
Mice (three wild-type and three mutant) were injected intravenously with 0.2 ml of 1% Evans blue on day 6, shortly before death of the wild-type mice. One hour later, mice were sacrificed and the staining of brain sections was assessed as an indicator of increased capillary permeability.
Light and Electron Microscopy
Mice were sacrificed by an intraperitoneal injection of nembutal and the aorta was cut. Brain was isolated and immersion-fixed in formaldehyde 5% in ethanol 70%. Frontal sections were prepared for ordinary histology. Venules within the cortex were examined and the endothelial cells and macrophages were counted. Endothelial cells were counted (>50/sections) as well as the number of macrophages and the sequestration was evaluated by the macrophage/endothelial ratio. Megakaryocytopoiesis was evaluated on sections of the spleen, by counting at a magnification of 400-fold, the number of megakaryocytes/microscopic field, as described previously.28
To examine the lung, the thorax was opened and the lung was fixed by intratracheal instillation of glutaraldehyde (5% in 0.1 mol/L cacodylate buffer) and processed for epon embedding. A section was taken in the left lobe across the hilus. Thin sections were prepared from two blocks/mouse taken from the parenchyma. Thin sections were examined with a Philips 400 electron microscope at 60 kV. Cells within alveolar capillaries were examined and red blood cells (RBCs), parasitized RBCs (pRBCs), platelets, and PMNs were counted. Approximately 100 RBCs were counted per each individual thin section. RBCs were used as a neutral indicator of blood stasis and sequestration was evaluated by the platelets/RBCs and PMN/RBC ratios.
Immunohistochemistry
Frozen tissue sections were immunostained as described elsewhere. Briefly, 5-µm sections were incubated overnight at room temperature with rat mAbs: anti-CD54 and anti-CD106 mAbs, as described previously,6 or anti-CD40 mAb FGK45.29 After washing, sections were incubated 1 hour with fluorescein isothiocyanate-labeled goat anti-rat IgG (Southern Biotechnology, Bioreba, Reinach, Switzerland).
Evaluation of TNF Serum Level
TNF-
was evaluated on the citrated plasma, using the mouse
enzyme-linked immunosorbent assay Dy 410 (R&D Systems, Inc,
Minneapolis, MN), which detects both free and receptor-bound TNF, with
a sensitivity of 12 pg/ml.
Evaluation of CD40L, CD40, TNF-, and CD54 mRNA Levels
TNF- (TNF),30
CD40L,31
CD40,32
and CD5433
mRNA levels were evaluated
on Northern blots, using 32P-labeled dUTP
riboprobes. Quantification of mRNA was performed using the Arcus II
system of quantification of scanned gels (Agfa, Mortsel, Belgium) with
the Image Quant Analysis (Molecular Dynamics, Sunnyvale, CA)
integration. The values were normalized to the 18S RNA and the amount
of mRNA was expressed in arbitrary units.34
Statistical Evaluation
Significance analysis of survival curves was determined by Fisher’s exact test. Groups of values were compared using Student’s t-test or the nonparametric Mann and Whitney test.35
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Infection with PbA results, in susceptible mice, in a lethal
complication 7 to 9 days after infection.8
More than 90%
of the infected +/+ mice were dead on day 8 after infection, while the
CD40-/- (Figure 1A)
or the CD40L-/-
(not shown) did survive beyond day 12. A few hours before death, mice
became hypothermic and comatose, and because a temperature below 33°C
is invariably followed by death, this criteria was used as an end point
in the majority of the experiments for ethical reasons. Thus,
hypothermia was evident on days 7 to 8 after infection in infected +/+,
but absent or markedly attenuated in CD40-/-, CD40L-/- mice, as
well as in +/+ mice treated with anti-CD40L mAbs (Figure 1, B and C)
.
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A breakdown of the blood-brain barrier, detected by a leakage of
Evans blue, as well as an increase of the vascular permeability in
other organs, such as the lung, are evident during PbA-induced
SM.3,6
A marked increase of the leak was evident in
infected +/+, but not in CD40-/- mice (Figure 2)
.
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Parasitemia was similar in +/+, CD40-/- and CD40L-/- mice
until day 8 after infection when the +/+ died (Figure 3)
while in the CD40-/- and CD40L-/-
mice, parasitemia kept increasing the following days. Mortality was
therefore not related to difference in parasitemia.
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Splenomegaly after PbA Infection
Seven days after infection, the spleen increased approximately
threefold in size, and this was evident in +/+ as well as in CD40-/-
mice, whereas in CD40L-/- mice, the splenomegaly was less pronounced
(Figure 4)
. In contrast, the size of the
lymph nodes did not change significantly in +/+ infected mice (not
shown).
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SM is associated with a severe thrombocytopenia because of a
reduced platelet life span.36
On the 7th day after
infection, +/+ mice were severely thrombocytopenic, but
thrombocytopenia was attenuated significantly, but to a modest extent,
in CD40-/- mice and more markedly in CD40L-/- (Figure 5A)
. Thrombocytopenia was also
significantly attenuated in +/+ mice treated with the anti-CD40L mAb,
compared to those treated with the control IgG (Figure 5B)
.
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PbA-induced SM is associated with the sequestration of various
cell types, notably in the brain and lung. In the brain, macrophage
sequestration was evident in the cortical venules of infected +/+ mice
and, as seen in Figure 6
, this was
markedly reduced in infected CD40-/- or CD40L-/- mice. In mice
treated with the anti-CD40L mAb, the modification of sequestration was
less conclusive. In some mice, sequestration was decreased, whereas in
others, it was modified by an increase of lymphocyte sequestration
together with macrophages. Sequestration of PMNs is not known to occur
in the cerebral blood vessels of PbA-infected mice and was not observed
in the present study (not shown). pRBCs are present in the brain
microcirculation and were sometimes observed in contact with the
venular endothelium by transmission electron microscopy (not shown), in
accord with a recent report.37
However, when the
percentage of pRBCs in the brain venules and capillaries was evaluated
by transmission electron microscopy, it was not higher than in the
systemic circulation, in contrast to what was observed in alveolar
capillaries (see below), therefore arguing against a selective
retention of pRBCs in brain microcirculation. Microhemorrhages are
common in PbA-infected CBA mice,5
whereas in contrast,
these are rare in mice of C57BL/6 background, and were therefore not
evaluated in the present experiments.
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, and Figure 8A
). Similar results were obtained in
three infected CD40L-/- mice studied (not shown). Using blood smears,
it has been reported that the percentage of pRBCs is higher in the lung
than in the peripheral blood.7
With the present method, we
also observed a higher percentage of pRBCs in the alveolar capillaries
than in the large blood vessels of the lung, indicating a selective
adhesion of pRBCs in alveolar capillaries (Figure 7C
and Figure 8B
).
This was similar in +/+ and CD40-/- infected mice (Figure 8B)
, as
well as in CD40L-/- mice (not shown). In CD54-/-, in contrast to
CD40-/-, no difference between the percentage of pRBCs in large blood
vessels and alveolar capillaries was observed (Figure 8B)
. These
observations indicate that the sequestration of pRBCs in alveolar
capillaries requires CD54, but not CD40.
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We examined the mRNA levels in the brain, lung, and spleen of
controls or PbA-infected mice (Figure 9)
.
The CD40 mRNA levels were significantly increased in the brain and lung
of infected mice, compared to noninfected mice (Figure 9)
. By
histochemistry, a weak staining of the cortical venules was observed
(not shown).
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.
However, taking into account the approximately threefold increase of
the spleen size, it is likely that the amount of CD40L mRNA per spleen
is increased. These observations suggest an increase of CD40L
production in infected mice. Expression of TNF, CD54, CD106, and CD31
TNF mRNA levels were significantly elevated in the brain, lung,
and spleen of infected +/+ mice. Surprisingly, in the lung and spleen,
the mRNA levels were significantly higher in CD40-/- or in CD40L-/-
than in +/+ mice RNA (Figure 10)
. In
the brain in contrast, TNF mRNA levels were increased in CD40-/- and
abrogated in CD40L-/- (Figure 10)
. In mice treated with the
anti-CD40L mAb, the TNF mRNA levels in the brain, lung, and spleen
showed similar trends to those seen in CD40L-/- (not shown).
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Expression of CD54 was evaluated both at the mRNA level and by
histochemistry. In the brain, the infection is known to increase the
CD54 mRNA levels as well as the expression of CD54 in venules. CD54
mRNA levels were slightly and similarly decreased in both CD40-/- and
CD40L-/- infected mice, compared to +/+ (Figure 10)
. By
histochemistry of brain section, CD54 was clearly increased in venules
of infected +/+ compared to controls. Expression of CD54 was also
increased in infected CD40-/- mice, without obvious difference with
infected +/+ mice (not shown).
In the lung, CD54 mRNA levels were slightly but not significantly
increased by infection in +/+ mice. Surprisingly, in infected CD40-/-
or CD40L-/- mice the levels were significantly different from the
infected +/+ but in opposite direction, markedly reduced in CD40-/-,
and significantly increased in CD40L-/- mice (Figure 10)
. In the
spleen, the modifications were similar to those seen in the lung
(Figure 10)
. Expression of CD106 in brain venules was increased in both
infected +/+ and CD40-/- mice, as seen by histochemistry, without
detectable difference between them (not shown).
CD31 is present in a variety of cells, including endothelial cells and platelets. No important modifications in the distribution of PECAM were evident in +/+ mice, infected or not or in infected CD40-/- mice (not shown).
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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PbA infection resulted in an approximately threefold increase in the
spleen size in 7 days,38
which was evident in +/+ and in
CD40-/-, but less pronounced in CD40L-/- mice. The splenomegaly
might contribute to the severity of the disease because the mortality
is prevented by splenectomy.39
In humans,
lymphoproliferative diseases have been reported to be associated with
an increase of the amount of CD40L in plasma.40,41
We were
unable to measure the CD40L plasma levels in infected mice, but the
evaluation of the mRNA levels in tissue showed an increased level in
the lung, and an increased level per spleen if the spleen size (Figure 4)
is taken into account. Expression of the cell receptor CD40, judged
from its mRNA was increased in the brain and lung, observations that
argue for an increase of CD40L-CD40 signaling during PbA infection.
TNF is believed to play a central role in many of the alterations
observed during SM, including the mortality. Because CD40/CD40L
deficiency is reported to disrupt cell-mediated immunity, it was
obviously of importance to evaluate whether the absence of CD40-CD40L
signaling decreases TNF production. In PbA-infected mice, we observed
similar TNF plasma levels in +/+, CD40-/-, and CD40L-/- mice.
Accordingly, TNF mRNA levels in the spleen and lung of infected
CD40-/- or CD40L-/- mice were not decreased and were even
significantly higher in CD40L-/- than in +/+ mice. These observations
indicate that absence of CD40 or CD40L did not markedly affect systemic
TNF production and that the blockade of the events leading to SM
resulting from the abrogation of CD40-CD40L signaling must be exerted
downstream of TNF production. This interpretation is also in accord
with the fact that the anti-CD40L mAb did prevent the occurrence of SM,
even when given on day 6 after infection, ie, immediately before the
effector phase of the disease. Surprisingly in the brain, TNF mRNA
levels were modified in opposite direction in CD40-/- or CD40L-/-
mice and almost abrogated in the latter. This observation suggests that
CD40L might exert a specific influence on the production of TNF in the
brain, independent of CD40. This discrepancy between the cerebral TNF
mRNA levels in the brain seen in CD40 and CD40L also indicates firstly,
that TNF mRNA levels in the brain are not correlated with morbidity,
(ie, body temperature) because CD40- and CD40L-deficient mice were not
sick, but with high or low cerebral TNF mRNA levels, respectively, and
secondly, that TNF mRNA levels in the brain are not correlated with
macrophage sequestration, because this was similarly reduced in
CD40-/- and CD40L-/- mice (Figure 10)
.
One of the effects of TNF is to increase the expression of adhesion molecules on endothelial cells, an activity shared with IL-1.25,42 Published reports, based on response of endothelial cells in vitro, suggest that CD40 signaling might also contribute to increasing the expression of adhesion molecules CD54 or CD106.25 In the present model, the role of CD40 and CD40L in the expression of CD54 was not identical and also organ-specific, ie, in the lung and spleen, CD54 expression was CD40-dependent but CD40L-independent. Because in these organs, the CD54 mRNA levels were significantly higher in CD40L-/- than in +/+ mice, this suggests that CD40L can also inhibit the expression of CD54. In the brain, in contrast to lung and spleen, expression of CD54 was decreased to a modest but similar extent in both CD40-/- and CD40L-/- mice, an observation compatible with the possibility that expression of CD54 in the brain contributes to the mortality of SM.11 The increased expression of CD54 in the brain of infected CD40L-/-, whose cerebral TNF mRNA was almost abrogated, indicates that circulating, but not cerebral, TNF is sufficient to up-regulate CD54 in cerebral venules.
Both CD40 and CD40L deficiency markedly decreased the sequestration of
macrophages in brain venules and alveolar capillaries. Because the
modest down-regulation of CD54 does not seem to be a sufficient
explanation, these observations raise the possibility that macrophages
bearing CD40L might bind to the endothelial CD40, although we could not
detect a significant expression of CD40 in cerebral venules by
histochemistry, in accord to what was observed in a study of multiple
sclerosis.43
Nevertheless, the decrease of macrophage
sequestration in the brain and lung of CD40-/- or CD40L-/- does
correlate well with the decreased mortality. Sequestration of pRBCs as
a possible pathogenic mechanism of PbA infection has been primarily
overlooked so far, because its occurrence in the brain microcirculation
has not been clearly demonstrated. pRBCs are indeed present in the
brain microcirculation,37
but in our experiments performed
with parasitemia <10%, we did not detect a significantly higher
parasitemia in the brain microcirculation than in large blood vessels,
arguing against a specific retention of pRBCs in the brain. In
contrast, parasitemia was significantly higher in the alveolar
capillaries than in the major blood vessels of the lung (Figure 8A)
,
arguing for a specific retention of pRBCs in the alveolar capillaries.
In addition, pRBCs arrested in the alveolar capillaries are the most
mature pRBCs (Figure 7C)
, in accord to what has been reported for other
rodent parasites.6,7
It has been suggested that pRBCs
might contribute to the severity of SM by interfering with the
microcirculation and/or by the release of toxic molecules damaging the
downstream circulation.44
pRBC sequestration was
significantly increased in alveolar capillaries of CD40-/- or
CD40L-/- mice (Figure 8B)
, indicating that it does not require
CD40-CD40L signaling, whereas it was not evident in CD54-/- mice, as
we reported previously.6
Because pRBCs sequestration was
obvious in CD40-/- or CD40L-/- mice that were otherwise not sick,
these results argue that pRBC sequestration is not sufficient to induce
alone the manifestations of SM. Sequestration of PMNs has also been
reported to contribute to the mortality of SM,5
but the
present results indicate that PMN sequestration, which was evident in
infected CD40-/- mice and is therefore CD40-independent, is also not
sufficient to elicit alone the complications of SM.
SM, as are other systemic inflammatory disorders such as endotoxemia or acute respiratory distress syndrome, is associated with a thrombocytopenia, because of a drastic reduction of platelet life span.45 Platelet loss might occur by platelet adhesion in the microcirculation, as it has been reported46 and/or by the fragmentation of platelets in plasma and the generation of microparticles.47 Both processes might influence the course of the disease, by the release, local or systemic, of platelet factors. In our experiments, blockade of CD40L by either genetic disruption or mAb did significantly attenuate thrombocytopenia, whereas the effect of CD40 deficiency was also significant but less pronounced. These results indicate that CD40L, whose production increases during infection, as discussed above, is an important effector of the PbA-induced thrombocytopenia. A significant contribution of CD40-CD40L in malaria-induced thrombocytopenia is in accord with a recent observation indicating that platelets bear CD40 and CD40L and that CD40L-mediated platelet activation can result in vascular damage.22 Because injection of TNF can induce a thrombocytopenia48 and because the thrombocytopenia of SM can be attenuated by anti-TNF (unpublished observation) as well as with anti-CD40L antibody, these observations indicate that both TNF and CD40L contribute to the platelet loss of SM.48
Interestingly CD40, as well as CD54 deficiency,6 did decrease both platelet loss and breakdown of the blood-brain barrier. Pathogenesis of vascular leak in the brain,49 as well as its possible role in the mortality are poorly understood. This vascular leak is evident in cortex and white matter and interests therefore most likely capillaries, unlike macrophage sequestration that is limited to venules. In the retina, leak seems to start in venules, and extends subsequently to capillaries.16 Because in other territories TNF can induce a vascular leak,50,51 it is possible that TNF contributes to the vascular leak of SM. However, the vascular leak was absent in CD40-/- mice despite elevated TNF plasma levels or elevated cerebral TNF mRNA levels, indicating that this effect of TNF might require the presence of CD40.
Thus, CD40-CD40L plays a crucial role in the mortality induced by PbA infection and the present results indicate that CD40-CD40L is necessary for the breakdown of the blood-brain barrier, macrophage sequestration, and platelet consumption associated with SM.
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Footnotes |
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This study was supported by grant no. 31-56839.99 from the Swiss National Science Foundation.
Accepted for publication April 30, 2001.
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References |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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