K . Yasojima et al. Brain Research 887 2000 80 –89
83
cent substrate Pierce Chemical Co., Rockford, IL. After containing 0.6 nickel ammonium sulfate and 0.001
draining, the membranes were covered in clear plastic H O in 0.05 Tris–HCl buffer, pH 7.6. When a dark blue
2 2
wrapping and exposed to X-ray film Hyperfilm ECL, color developed, sections were washed, mounted on glass
Amersham Life Science, UK for 0.3 to 2 min, depending slides and coverslipped with Entellan.
on the strength of the signal. The primary antibodies against human CRP were rabbit anti-CRP DAKO, 1:500,
sheep anti-CRP WAKO, 1:1000, mouse monoclonal anti-
3. Results
CRP 6804 Medix Biochimica, 1:100, and mouse mono- clonals anti-CRP-1 and anti-CRP-4 kind gifts of Dr
3.1. Relative mRNA levels Gregory Lee, each at 1:100. Each antibody gave a single
band on tissue extracts comigrating with authentic CRP The results of reverse transcriptase–polymerase chain
standard. The primary antibody against human AP was reaction RT–PCR analysis on the liver and five brain
rabbit anti-AP Calbiochem, 1:3000. The secondary anti- regions from AD and neurologically normal cases are
bodies were from Sigma 1:8000. shown in Fig. 1 and Table 1 see Materials and methods
for details. Fig. 1 shows polaroid photographs of typical 2.5. Immunohistochemistry
ethidium bromide-stained gels. Fig. 1a is for CRP mRNA in a normal case, and Fig. 1b for an AD case. Signals were
Control cases without neurological symptoms or neuro- obtained from all brain areas and from liver in both normal
pathological findings and confirmed AD cases were select- ed for study from our brain bank at the University of
British Columbia. Brain tissues for routine immunocytoch- emistry had been fixed in 4 paraformaldehyde and, after
3 to 4 days, transferred to a 15 buffered sucrose maintenance solution. Some blocks were further embedded
in paraffin after fixation. Other tissue blocks were fixed in 70 ethanol in saline for 1 day before transfer to the
sucrose maintenance solution. The monoclonal antibodies to CRP required such fixation to give positive immuno-
staining. Sections from paraformaldehyde and ethanol fixed tissues were cut at 30 mm on a freezing microtome.
The paraffin blocks were cut at 10–15 mm on a standard microtome. The paraffin sections were deparaffinized in a
series of xylene and ethanol concentrations. All sections were incubated free floating for immunocytochemistry as
previously reported [1,32,37].
Sections for processing with the rabbit polyclonal anti- human CRP antibody DAKO were treated for 1–5 min
with concentrated formic acid. Such pretreatment greatly enhanced the immunohistochemical staining [6,15]. These
and other sections not exposed to formic acid were treated for 30 min with 0.5 H O solution in 0.01 M phosphate-
2 2
buffered saline, pH 7.4, containing 0.3 Triton X-100 PBS–T. They were transferred into 5 skim milk in
PBS–T for 30 min and incubated for 72 h at 48C or
Fig. 1. Representative polaroid photomicrographs of ethidium bromide
overnight at room temperature with one of the anti-human
stained electrophoretic gels of RT–PCR products of CRP a, b and AP c,
primary antibodies.
These were
anti-CRP DAKO,
d RT–PCR products. Gels from an AD case b, d are compared with a normal case a, c. Lane 1, size markers; lane 2, hippocampus; lane 3,
1:5000; anti-CRP 5G4 kind gift of Dr W.K. Lagrand,
midtemporal gyrus; lane 4, midfrontal gyrus; lane 5, motor cortex; lane 6,
Amsterdam, MAB, 1:1000; anti-CRP 6804 Medix Bio-
cerebellum; lane 7, liver. A single band of 440 bp was obtained for the
chimica, MAB, 1:100; anti-CRP 6405 Medix Bio-
CRP product and 309 bp for the AP product see Materials and methods
chimica, MAB, 1:500; and rabbit anti-AP Calbiochem,
for details. Notice in the normal case for CRP that only weak bands were
1:3000. Sections were next treated with appropriate
obtained from brain areas, with a much stronger band from liver. By contrast, in the AD case, stronger bands were obtained from brain areas,
biotinylated secondary antibodies for 2 h at room tempera-
especially the heavily affected hippocampus and midtemporal gyrus
ture, followed by incubation in avidin–biotinylated horse-
lanes 1 and 2, than from liver in the same case. For AP, easily
radish peroxidase complex ABC Elite, Vector Labs for 1
detectable bands were obtained from all areas of the normal c and AD
h at room temperature. Peroxidase labeling was visualized
d cases, with more prominent bands from the AD case. However, much
by incubation in 0.01 3,3-diaminobenzidine Sigma
stronger bands were detected from the livers of these cases.
84 K
Table 1 Relative levels mean6S.E.M. of the mRNAs for amyloid P, C-reactive
protein CRP and the housekeeping gene cyclophilin CP in five brain
a
regions and liver in AD and controls Tissue
Amyloid P CRP
CP Midtemporal-AD
41.0462.37 51.9162.21
84.3560.70 Control
17.1661.19 2.1360.28
85.5060.68 Hippocampus-AD
43.2861.96 58.6662.43
85.7561.07 Control
21.0461.15 3.1360.66
85.0660.60 Midfrontal-AD
39.4462.37 30.3462.18
85.9660.92 Control
17.9662.24 3.7260.72
84.9960.63 Motor-AD
40.8463.04 16.1163.42
84.9160.80 Control
18.4461.27 3.1560.40
85.5560.91 Cerebellum-AD
32.4863.10 9.3762.92
85.2761.02 Control
19.2061.40 3.8260.42
86.4360.76 Liver-AD
146.163.2 5.8861.31
97.0660.54 Control
148.067.6 7.4360.73
96.9160.58
a
P,0.001, P,0.01, P,0.05 for difference between AD and control cases; all corrected by Holm’s stepdown procedure [13] for
multiple analyses. The calculations for CRP and AP mRNA levels used data normalized to the cyclophilin values.
and AD cases. In normal brain, however, these signals were very faint, but in AD cases, as illustrated in Fig. 1b,
intense signals were observed in the hippocampus and midtemporal gyrus. Intermediate signals were seen in the
midfrontal and motor cortices, with only a weak signal in the cerebellum. As illustrated in the figure, the AD brain
areas showed a stronger signal than the liver.
Figs. 1c and 1d show comparable data for AP. Fig. 1c shows a polaroid photograph of a typical ethidium bromide
gel for a normal case and Fig. 1d for an AD case. Signals for AP mRNA were clearly visible from all areas in the
normal case and in AD they were not increased as dramatically as those for CRP mRNA. Moreover, the
signals from liver were much stronger than from any of the brain areas.
Fig. 2 illustrates the lack of effect of PMD and agonist
Fig. 2. Bar graphs showing the mean6S.E.M. of the mRNAs in arbitrary
causes of death on the mRNA levels of the constituents
OD units for cyclophilin A, CRP B and AP C for the seven AD and
under study. The housekeeping gene cyclophilin gave
eight control cases. The eight controls were divided into four cases with a
almost identical values in every region of brain in AD
post-mortem delay of 20 h or less average 14.2 h and four with a
cases, controls with short PMD and controls with long
post-mortem delay of 24 h or more average 48 h.
PMD. For CRP and AP, the levels varied widely according to disease and area but again values for short-term PMD
were almost identical with those for long-term PMD. cerebellum. These differences were highly significant
Accordingly, the eight control cases were combined for except for the cerebellum P50.065 which had a rela-
overall statistical analysis. tively large S.E.M. In the liver, there was no significant
Table 1 summarizes the semiquantitative mRNA values difference between AD and control values. Surprisingly,
in the AD and the combined control cases for CRP, AP and the liver mRNA levels were lower than in all areas
the housekeeping gene cyclophilin. Data were analyzed measured of AD brain.
uncorrected and normalized to the cyclophilin value ob- With AP mRNA, the AD cases showed significantly
tained in a parallel amplification. Since the cyclophilin higher mRNA values compared with controls in each brain
values were almost constant in all areas of AD and control area
investigated, although
the increases
were less
brains Fig. 2A, the significances were identical. The CRP dramatic than for CRP. The level was 2.4-fold higher in the
mRNA showed a 24.3-fold increase in AD cases compared midtemporal gyrus, 2.1-fold in the hippocampus, 2.2-fold
with controls in the midtemporal gyrus. The increase was in both the midfrontal and motor cortices, and 1.7-fold in
18.7-fold in the hippocampus, 8.2-fold in the midfrontal the cerebellum. Again, the liver values were no different
gyrus, 5.2-fold in the motor cortex and 2.4-fold in the between AD and control cases. But in the case of AP, the
K . Yasojima et al. Brain Research 887 2000 80 –89
85
liver levels were approximately 3.6-fold higher than in AD Western blots of serum up to a concentration of 10 mg
brain. The stronger signal for AP compared with CRP in of total protein failed to show a detectable band for either
liver is consistent with their differing base values in CRP or AP data not shown. Since this level of serum
normal serum. These are typically of the order of 20–30 protein is well beyond the residual level that would remain
mg ml for AP [28] but usually less than 2 for CRP [8], in the volume of brain tested [4], it can be concluded that
indicating a higher level of liver AP production compared the Western blots of Fig. 3 reflect proteins derived from
with CRP. brain substance itself. The relative intensity of the bands in
Fig. 3 compared with authentic standards indicate a level 3.2. Western blotting
of AP in normal hippocampus of approximately 0.6 mg mg protein and in AD a level of about 1 mg mg protein.
Examples of Western blotting results are shown in Fig. CRP was below detection levels in normal hippocampus,
3a for CRP and Fig. 3b for AP. Protein extracts of the but in AD it was approximately 1.4 mg mg of protein.
hippocampus in typical AD and control cases were com- These Western blots are consistent with the mRNA data of
pared, as well as liver extracts from the same cases. Table 1, indicating that, for the areas examined, the
Extracts were run in parallel with authentic protein stan- mRNAs were being translated into their protein products in
dards. Identical results were obtained with three commer- approximate proportion to their relative mRNA levels,
cial anti-CRP antibodies; a rabbit polyclonal DAKO, a with normal levels for CRP in brain being below the limits
sheep polyclonal WAKO and a murine monoclonal of Western blot detection.
6804, Medix Biochmica and two other murine mono- clonals CRP-1 and CRP-4, gifts of Dr G. Lee. There was
3.3. In situ hybridization no detectable band for CRP from normal hippocampus but
a single strong band was observed in AD, with a weaker Strong in situ hybridization signals for both CRP and
band being seen in liver. The tissue extracts migrated AP mRNAs were detected over neurons in the AD
equally with authentic CRP standard ca. 25 kDa. Fig. 3b hippocampus entorhinal cortex and adjacent temporal
shows a comparable analysis for AP with a polyclonal neocortex. The distribution of positive signals was the
rabbit antibody Calbiochem. A single AP band was same for both pentraxins. Signals were mostly present in
picked up from normal hippocampus, with a stronger band pyramidal cells although granule cells of the dentate gyrus
from AD hippocampus, and a considerably more intense were strongly positive for both pentraxins. Typical results
band from liver. All bands migrated equally with the are shown in Fig. 4. Fig. 4a is a photomicrograph of the
authentic AP standard ca. 25 kDa. CA4 region of the hippocampus in an AD case stained
with the anti-sense CRP probe. A strong signal is observed over many pyramidal neurons. Fig. 4b is an adjacent
section stained with the sense probe. No signal is observ- able. Fig. 4c is a photomicrograph of the CA4 hippocam-
pal region in an AD case stained with the anti-sense AP probe. Again, a strong signal is observable over many
pyramidal neurons. Fig. 4d is an adjacent section stained with the AP sense probe, with no signal being observable.
Figs. 4e and g illustrate hybridization of granule cells in the dentate gyrus with the CRP and AP antisense probes
respectively. Figs. 4f and h illustrate hybridization in the hippocampal CA3 and CA1 pyramidal cells with CRP and
AP antisense probes, respectively, and Figs. 4i and j hybridization in the entorhinal cortex and adjacent tempo-
ral neocortex using CRP and AP antisense probes. No signal was detected in any of these regions using the CRP
and AP sense probes. Very weak signals in layer 1 of the entorhinal and neocortex may indicate some mRNA pro-
duction by glial cells.
Fig. 3. Western blots of the hippocampus of a normal lane 1, AD lane
3.4. Immunohistochemistry
2 and AD liver lane 3 compare with authentic standards lane 4, 0.5 mg for CRP a and AP b. Single bands were detected co-migrating
Largely comparable immunohistochemical staining was
with the standards in each case ca. 25 kDa. Notice the much stronger
observed with antibodies to CRP and AP, but there were
bands for both CRP and AP in the AD case compared with the control.
some differences. All antibodies tested showed intense
Detection was with the anti-CRP-4 MAB but similar data were obtained with four other antibodies see Materials and methods for details.
immunostaining of eNFTs and some iNFTs for both
86 K
Fig. 4. In situ hybridization for CRP anti-sense a, e, f, i, CRP sense b, AP antisense c, g, h, j, and AP sense d see Materials and methods for details. Strong signals are seen over pyramidal neurons in hippocampus CA4 for both the CRP a and AP c anti-sense probes, but no signals are seen for
the CRP b and AP d sense probes. Dentate granule cells also give strong signals for CRP e and AP g. Hippocampal CA1 pyramidal neurons can be seen to be positive for the CRP f and AP h antisense probes. Pyramidal neurons of the entorhinal cortex are also positive with the CRP antisense probe
i, and those of the adjacent temporal cortex with the AP antisense probe j. Sense probes in all of these areas gave no signal.
pentraxins Fig. 5a, d, e. There was also positive immuno- extracellular amyloid material Fig. 5g. These immuno-
staining for both pentraxins in some normal appearing histochemical results confirm previous reports of CRP
pyramidal neurons Fig. 5b and f although most were [6,15] immunostaining of NFTs and senile plaque elements
unstained. The polyclonal CRP antibody stained some in formic acid-treated sections, and extend them to include
dystrophic neurites, especially in association with senile some normal appearing neurons. Similarly, the AP im-
plaques Fig. 5c. The AP antibody stained the plaques munostaining confirmed earlier reports of immunostaining
more homogeneously, suggesting a strong association with of NFTs and senile plaques [3,7,14,32]. These immuno-
K . Yasojima et al. Brain Research 887 2000 80 –89
87
employed coupled with higher sensitivity primers. The signal from liver is considerably stronger than from brain,
and insensitive techniques could easily miss tissues other than the liver.
The data show that, at least for the mRNAs being studied here, there was no evidence of post-mortem
deterioration for periods up to 96 h after death. Such stability in tissues that have not been frozen has previously
been reported by us [41,42], as well as others [16,26]. However, the methods reported here would only detect
cleavage within the regions amplified by RT–PCR and would be insensitive to cleavage at other sites.
There have been many previous reports on the immuno- histochemical detection of AP in fixed brain tissue. But
CRP has been much more difficult to identify even though it is easily detected by Western blot. Accordingly, we
tested two polyclonal and three monoclonal antibodies for their ability to detect CRP at the cellular level. The three
monoclonal antibodies recognized CRP only on ethanol- fixed tissue, and one polyclonal only on fixed tissue
following formic acid pretreatment. The reasons are un- known.
CRP and AP have relatively high turnover rates. The half-life of CRP in serum is about 19 h [39], with
comparable values being obtained for serum AP [10].
123
Scintigraphy with I-labeled AP showed uptake and
retention of AP in peripheral amyloid deposits [10]. However, there was no measurable uptake and labeling of
AD brain amyloid deposits [22]. Nevertheless, AP has been detected in normal CSF, with upregulation in AD
[11,18]. This is consistent with serum AP failing to cross
Fig. 5. Immunohistochemistry for CRP a–d and AP e–g. Sections were formic acid treated and stained with polyclonal anti-CRP antibody
the blood–brain barrier and with production of AP in brain
a–c, ethanol-fixed and treated with 5G4 MAB d, or paraffin embedded
accounting for much of the CSF content. There was also
and treated with the polyclonal anti-AP antibody e–g see Materials and
123
no detectable uptake of I-labeled CRP into brain [39],
methods for details. Photomicrographs show strong CRP immunostaining
although CRP has been detected in normal CSF, with
of eNFTs a, of neurons b, plaques c and iNFTs d. The senile plaque
dramatic upregulation in bacterial meningitis [34]. Again,
in c shows granular staining for CRP, probably in dystrophic nerve endings. Dystrophic neurites are also visible in a and b arrowheads. The
these results are consistent with brain production of CRP
AP antibody stained tangles e, normal appearing neurons f and senile
accounting for much of its presence in normal CSF.
plaques g. Note, however, the different appearance of senile plaque
CRP has previously been reported to be produced by
staining of AP compared with CRP. The AP immunostaining g is
alveolar macrophages in addition to hepatocytes [5], but
stronger and appears to be localized to the extracellular amyloid material.
AP has been reported only to be produced by hepatocytes
Scale bar in g applies to a–g550 mm.
[17]. In results to be reported elsewhere, we have found mRNAs for both CRP and AP in lung, heart, arteries,
kidney and spleen, so many types of cells in addition to histochemical results are consistent with the proteins being
hepatocytes, macrophages and neurons may produce these generated primarily within neurons and then becoming
pentraxins. deposited on extracellular lesions.
Numerous functions have been associated with CRP, all of them related to inflammatory processes and host defense
[36]. But CRP may not always play a protective role. CRP
4. Discussion binds to phosphocholine in complement-damaged cell
