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Technical Advances |
From the Departments of Pathology*
and Internal
Medicine,
and the Institute for Anatomy and
Cell Biology,
Justus-Liebig-University,
Giessen, Germany
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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nebulization. Whereas AMs from
the left lung were directly harvested for mRNA extraction by
bronchoalveolar lavage, tissue sections of the right lung were
stained with an optimized immunofluorescence protocol detecting AMs.
Fifteen AM profiles per sample were picked by laser-assisted sampling
technique. Normalizing to a standard gene, nitric oxide
synthase II (NOSII) and tumor necrosis factor (TNF)-
mRNA were
quantified by real-time reverse transcriptase-polymerase chain
reaction. In stimulated lungs, the percentage of picked samples
positive for NOSII or TNF- mRNA increased significantly.
Moreover, a marked increase in the ratio of target gene mRNA to
standard gene mRNA was noted for both NOSII and TNF- in picked AMs
from stimulated lungs, which matched very well the increase
detected in the lavaged AMs undergoing direct RNA extraction.
Thus, when using an optimized protocol for
immunofluorescence, this approach may be reliably combined with
laser-assisted cell picking and real-time mRNA quantitation in a few
immunohistochemically characterized cell profiles within complex
tissues.
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Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Using an ultraviolet laser microdissection system, mRNA analysis was shown to be possible even in a few cell profiles originating from formalin-fixed routine material2 or cryosections.3 Additionally, this technique allows identification of distinct splicing variants within a few cell profiles.4-6 Moreover, our group as well as other workers have demonstrated a reliable mRNA quantitation in microdissected samples from hemalaun- or hemalaun/eosin-stained tissue sections.7,8 Immunostaining and microdissection for mRNA analysis were found to be possible for several hundred cell profiles,9 for single dispersed, but intact cells,10 and for single cell profiles further processed by an RNA amplification step.11 However, we observed a decrease in efficiency rates for mRNA detection (percentage of samples positive for the respective mRNA) when routine immunostaining was applied.12 In an attempt to optimize the technique with respect to mRNA recovery, we showed that short-term formalin fixation, utmost reduction of antibody incubation times, application of immunofluorescence, and digestion with proteinase K forwarded the best results when applied to oligocellular clusters.
In the present study, we combined immunolabeling, microdissection, and
picking of a few stained cell profiles and mRNA quantitation by
real-time RT-PCR.13,14
In ex vivo ventilated
and perfused rat lungs, aerosolization of lipopolysaccharide (LPS) and
interferon- (IFN-) was used to up-regulate alveolar macrophage
(AM) nitric oxide synthase II (NOSII) and tumor necrosis factor-
(TNF-) mRNA levels. AMs were then obtained by conventional
bronchoalveolar lavage. Alternatively, they were stained within tissue
sections of the lung by use of the indirect immunofluorescence
technique, and 
15 cell profiles per sample were isolated for mRNA
quantitation. Relative quantitation was performed by normalizing NOSII
and TNF- mRNA to mRNA of the standard gene porphobilinogen deaminase
(PBGD). Thus, by comparing the data obtained by combined
immunofluorescence and cell picking to those from nonstained AMs
recovered by a standard procedure, validation of the technique was
obtained.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Male CD rats (Sprague-Dawley, 60 to 70 days old, 350 to 400 g
body weight; Charles River, Sulzfeld, Germany) were deeply anesthetized
with phenobarbital-Na. Lungs were isolated, ventilated, and ex
vivo-perfused with Krebs-Henseleit buffer in a closed perfusion
circuit as previously described.15
Measurement of
perfusion pressure, ventilation pressure, and organ weight was
performed continuously. The lungs had a homogeneous white appearance
without signs of hemostasis or edema formation and the pulmonary as
well as ventilation pressures were in the normal ranges. They were
isogravimetric during a steady-state period of 30 minutes. For
stimulation, an ultrasonic nebulizer was used afterward to aerosolize
75 µg of LPS and 1,000 U of IFN- in a volume of 5 ml into the
afferent limb of the ventilator circuit for 10 minutes. Experiments
were then continued under standard conditions for 6 hours. Control lung
did not undergo LPS/IFN- challenge. Certain differences in the
extent of mRNA up-regulation were seen among the stimulated lungs and
could most probably be because of individual variability of the organs
or smallest and nonsusceptible alterations of the experimental system.
For fixation, lungs were perfused with 4.5% formaldehyde solution (Roti-Histofix; Roth, Karlsruhe, Germany) for 15 minutes, followed by a rinsing step with Krebs-Henseleit buffer to remove the residual formalin. Finally, the left lung was lavaged, while the right lung was instilled with Tissue-Tek (Sakura Finetek, Zoeterwoude, The Netherlands) and snap-frozen in liquid nitrogen.
Bronchoalveolar Lavage
As detailed by Fink and colleagues,7 six aliquots of 3 ml of saline were instilled into the left lung and re-aspirated immediately. Cells were centrifuged at 300 x g, washed, and counted in a hemocytometer chamber. For in vitro stimulation, AM aliquots were transferred to plastic dishes and suspended in RPMI 1640 (Life Technologies, Paisley, Scotland) supplemented with 2% rat serum. Next, they were incubated at 37°C with 5% CO2. For mRNA extraction, AMs were directly transferred into 300 µl of lysis buffer (Dynal, Oslo, Norway).
In Vitro Stimulation
AMs were allowed to adhere for 2 hours. Afterward, medium was
replaced by medium supplemented with 10 µg of LPS and 1,000 U
IFN-. Cells were harvested after the respective incubation times at
37°C, 5% CO2, by removing the medium, lysed in
300 µl of lysis buffer (Dynal) and transferred into 1.5 ml reaction
tubes.
mRNA Extraction
Using the Dynabeads mRNA direct kit (Dynal), cell lysate was applied to magnetic separation. The mRNA was caught by attachment to oligo-dT fragments that are coupled to supermagnetic glass particles. Per sample, 100-µg beats were used. Isolated mRNA was finally solved in 2 x 10 µl of diethyl pyrocarbonate-treated H2O.
Immunofluorescence Labeling
Cryostat sections (8 µm) were mounted on
poly-L-lysine- (Sigma Aldrich, Deisenhofen, Germany)
covered glass slides and stored in acetone for 1 to 3 minutes.
Afterward, the murine primary monoclonal antibody ED-1 (specific for
rat macrophage/monocyte, 1:25; Serotec via Biozol Diagnostika, Eching,
Germany) was incubated for 5 to 8 minutes at room temperature, and
after shortly washing in buffer, incubation with fluorescein
isothiocyanate-conjugated goat anti-mouse immunoglobulin (1:100;
Organon Teknika, Durham, NC) at room temperature was performed for 5 to
8 minutes. Finally, sections were washed and immersed in 70 to 90%
ethanol until picking.
Immunoalkaline-Phosphatase Staining
Immunohistochemistry was performed by the alkaline-phosphatase
monoclonal anti-alkaline phosphatase (APAAP) technique, slightly
modified from the method of Cordell et al.16
Cryostat
sections (8 µm) were mounted on poly-L-lysine-covered
glass slides. To unmask the epitopes, sections were placed in a
solution of citric acid (2 µmol/L) and sodium citrate (9.1 µmol/L).
They were heated three times for 5 minutes in a microwave oven (600 W;
Bosch, Stuttgart, Germany) and cooled down to room temperature for 15
minutes. After storage in acetone for 10 minutes, they were incubated
with the primary antibodies rabbit polyclonal anti-NOSII (N-20, 1:250;
Santa Cruz Biotechnology, Heidelberg, Germany) or rabbit polyclonal
anti-TNF- (ICC-TNF-9B, 1:250; Innogenetics, Heiden, Germany) for 20
minutes. In a second incubation step for 20 minutes, monoclonal mouse
anti-rabbit immunoglobulin (1:500; Dako Diagnostika, Hamburg, Germany)
was applied, followed by incubation with rabbit anti-mouse
immunoglobulin (rabbit-"link," 1:40; Dako) and finally mouse-APAAP
complex (1:50; Dako), each at room temperature for 20 minutes. Second
and third antibodies were supplemented with pooled rat serum (1:750;
Sigma). Between these steps, samples were washed twice in Tris-buffered
saline (pH 7.5). Alkaline-phosphatase substrate reaction was performed
at pH 9.0 with new fuchsin (100 µg/ml) and levamisole (400 µg/ml)
for 25 minutes at room temperature. Finally, sections were
counterstained with hemalaun for 45 seconds.
Laser-Assisted Cell Picking, Proteinase K Digestion, and RT
Cell picking was performed as described in detail recently.5,12 Using the UV-laser Robot Microbeam (P.A.L.M., Bernried, Germany), a mercury vapor lamp was coupled to the epifluorescence illumination path. After immunofluorescence staining, AMs were selected by immunofluorescence microscopy. Every single AM localization was stored by the stage position, so that a precise return was possible. Afterward, bright-field illumination was switched on. The AMs were then identified by nearly closing the condenser. Adjacent tissue was removed by UV-laser photolysis under visual control. Finally, the AM profiles were isolated by a sterile syringe needle and transferred into a reaction tube containing 10 µl of first-strand-buffer. Samples with 15 AM profiles each were snap-frozen in liquid nitrogen and stored for further preparation. After a short thawing period, proteinase K (1 µl, 1 mg/ml; Sigma) was added to the samples and incubated for 1 hour at 53°C. Proteinase K as well as RNA were denaturated for 5 minutes at 95°C, and samples were stored on ice for another 5 minutes. To create identical conditions for picked samples and extracted mRNA (10 µl), they underwent denaturation and after cDNA synthesis in parallel using the same composition of the reagents. cDNA synthesis was performed using 2 µl MgCl2 (25 mmol/L), 2 µl GeneAmp 10x PCR buffer II (100 mmol/L Tris-HCl, pH 8.3, 500 mmol/L KCl), 1 µl dNTP (10 mmol/L each), 1 µl random hexamers (50 µmol/L), 0.5 µl RNase inhibitor (10 U), and 1 µl murine leukemia virus RT (50 U). Except for dNTP (Eurobio, Raunheim, Germany), all reagents were purchased from PE Biosystems (Weiterstadt, Germany). Samples were incubated at 20°C for 10 minutes and at 43°C for 60 minutes. Finally, the reaction was stopped by exposure to 99°C for 5 minutes.
Relative mRNA Quantitation
Real-time PCR is based on the 5' nuclease activity of
Taq polymerase for fragmentation of a dual-labeled
fluorogenic hybridization probe.13,14
Using the Sequence
Detection System 7700 (PE Biosystems), it was performed as recently
described in detail by Fink and colleagues.7
For relative
quantitation, the target gene was normalized to an internal standard
gene. Therefore, PBGD mRNA was used.5
This kind of
quantitation is calculated by the following equation:
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Using the mentioned primer/probe sets (Table 1)
, pilot experiments showed that
amplification efficiencies of PBGD, TNF-, and NOSII mRNA were noted
to be approximately equal and amounted to 0.95 ± 0.02.
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Twenty-five µl of Universal Master Mix (PE Biosystems), oligonucleotide primers (final concentration, 900 nmol/L), and hybridization probe (final concentration, 200 nmol/L) were added to an end volume of 50 µl. Cycling conditions were 95°C for 6 minutes, followed by 55 cycles of 95°C for 20 seconds, 61°C for 30 seconds, and 73°C for 30 seconds.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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were added. Maximal
up-regulation of NOSII occurred after 6 hours (Figure 1)
. In consequence, the isolated rat
lungs were ventilated and perfused for this time span.
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. Within hemalaun-stained tissue
sections of these lungs, PBGD mRNA was detected in 70% of the
samples containing 15 cell profiles or cell clusters with 15 nuclei.
Although this PBGD efficiency rate displayed virtually no variation
between different cells in the lung, NOSII mRNA was found in <10% of
the samples from bronchial epithelium and endothelium of both pulmonary
arteries and veins. Highest NOSII mRNA recovery in picked lung cells
was obtained for AMs (37.5%). In samples from alveolar septae
(including some monocytes/macrophages), the NOSII efficiency rate was
18.8%. According to these results, immunohistochemical staining of
lung tissue for NOSII detected predominantly AMs (Figure 3H)
.
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15 AM profiles per sample were picked (Figure 3, A–F)
, and
relative quantitation of NOSII and TNF- mRNA was obtained by
real-time RT-PCR.
Overall, 214 samples were analyzed from which 121 were positive for
PBGD mRNA (=56.5%; Table 2
). The mean
threshold cycle (CT) for PBGD mRNA varied only negligibly, thus
confirming the presence of similar mRNA amounts within all samples.
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mRNA was detected in 17.8% of the
samples, whereas NOSII mRNA was totally undetectable. In contrast,
41.5% of the samples from the stimulated lungs were found to express
TNF- mRNA and 33.8% NOSII mRNA, respectively. Thus, efficiency
rates of samples positive for TNF- and NOSII mRNA differed
significantly between control and stimulated lungs (NOSII,
P < 0.001; TNF-, P = 0.019;
Person’s chi-square test).
As a basis for the relative mRNA quantitation, the mean threshold cycle
for PBGD mRNA was calculated for each lung. The corresponding threshold
cycles of the target gene samples were substracted from mean PBGD CT
(
CT = CTPBGD -
CTtarget gene; given as
mean ± SEM). Thus, normalization to the nonregulated standard
gene PBGD was achieved. For lavaged and picked AMs, these CT values
are shown in Figure 4
(top). Calculating
a PCR efficiency of 0.95 ± 0.02 for the three investigated genes,
the values of relative mRNA expression were obtained by the formula
K·1.95CT (K =
constant). These values are given in Figure 4
(bottom).
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Not only the recovery of positive NOSII samples was shown to
differ significantly between control and stimulated lungs, the value of
relative NOSII mRNA expression in the positive samples also increased
significantly from control to stimulated lungs
(P < 0.001). In the control lung, NOSII mRNA
was not detected in either lavaged or in picked samples. In stimulated
lung case 1, 0.92 copies NOSII mRNA per copy PBGD mRNA was found for
lavaged AMs, and picked AM samples exhibited 0.56 ± 0.28 copies
NOSII mRNA per copy PBGD mRNA (Figure 4
, bottom). Relative NOSII
expression in lavaged AMs of stimulated lung case 2 amounted to 0.42
copies per copy PBGD mRNA, corresponding to 0.39 ± 0.09 copies in
picked AMs.
Relative TNF- mRNA Expression in Lavaged and Picked
Immunostained AMs
Relative TNF- mRNA expression in the control lung amounted to
0.17 copies per copy PBGD for lavaged AMs, and the picked AMs expressed
0.32 ± 0.05 copies TNF- mRNA. In stimulated lung case 1,
TNF- expression in lavage was found to be 4.68 copies per copy PBGD
mRNA, in the second stimulated lung 9.49. Corresponding expression in
picked samples amounted to 1.70 ± 0.67 copies and 2.13 ±
0.62 copies, respectively. Again, apart from the significant increase
in the percentage of positive TNF- mRNA samples in stimulated lungs,
the value of relative TNF- mRNA expression differed significantly
between the stimulated lungs and the control (P
< 0.001).
To confirm the results of mRNA quantitation, immunohistochemical
staining for NOSII and TNF- was performed in the control lung and
stimulated lungs. Within the stimulated lungs, NOSII was exclusively
stained in AMs. In the control lung, no staining was detectable (Figure 3, G and H)
. Additionally, immunohistochemical staining for TNF- was
performed. Again, only AMs of the stimulated lungs were labeled,
corresponding closely to the NOSII staining (data not shown).
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Because long-term immersion of tissue in aqueous media is deleterious
for RNA preservation,9
short-term indirect
immunofluorescence proved to provide the best mRNA efficiency
rates.12
Using ED-1 antibody to stain AMs, a specific
detection was obtained within 15 minutes. Unfortunately, the
additionally used anti-NOSII and anti-TNF- antibodies did not detect
their specific epitopes in formalin-fixed tissue. Pretreatment with
citrate buffer and microwave did indeed unmask these epitopes and
allowed specific staining. This, however, was shown to be
disadvantageous for nucleic acids and amplification.17
In
consequence, the suitability and shortest possible incubation time for
staining had to be tested for each primary antibody. Finally, the
influence of the fluorochrome conjugated to the secondary antibody on
the real-time PCR technology, based on fluorescence detection, must be
taken into consideration. In our experience, however, the combination
of fluorescein isothiocyanate-coupled secondary antibody and 6-carboxy
fluorescein or VIC-labeled PCR probes has never resulted in
interference with analysis or deviation of background measurement.
Although hemalaun staining of formalin-fixed tissue resulted in an
efficiency rate of 70% for PBGD in picked AMs, this was decreased
to a mean of 56.5% after immunofluorescence labeling. These rates
correspond well with our previous data.12
The loss might
be because of exogenous RNase activity introduced by the applied
antibodies or also to endogenous degradation mechanisms and elution,
despite the mild fixation. Additionally, the stimulation procedure
itself apparently influenced the efficiency rate of PBGD mRNA detection
in picked AMs (51.6% in stimulated as compared to 68.9% in control
lungs); the underlying reasons are currently still unknown.
Nevertheless, the mean CT for PBGD mRNA was substantially decreased
when compared to our previous study (37.15 versus 41.13),
underlining the beneficial effect of the newly introduced optimizing
steps.
Concerning the preferable type of tissue fixation, the best results in RT-PCR amplification products were obtained from cryosections fixed by a precipitating fixative like ethanol5,18 or methacarn19 in combination with a short routine staining (eg, hemalaun, hematoxylin and eosin). Unfortunately, in picked samples from organs fixed by ethanol, the RNA efficiency rate after the immunostaining procedure decreased considerably when compared to samples from organs fixed by the cross-linking fixative formalin.12
The mRNA analysis in picked AMs differs in many respects from mRNA
extracted from lavaged AMs. Whereas lavaged cells are intact with
integral cell membranes, picked cell profiles are sectioned
and thus lose a considerable portion of their cytoplasm and protecting
membrane. In addition, they have to pass the immunostaining procedure,
storage in alcohol, lasering, and picking. Nevertheless, these few
picked immunostained cell profiles are sufficient to reliably detect
the up-regulation of both NOSII and TNF- in response to inflammatory
challenge of the intact lungs. This was demonstrated first by a
significant increase in the percentage of both NOSII- and
TNF--positive samples as compared to samples from control lungs.
Second, it was shown by a manifold increase in target gene mRNA copies
(normalized to PBGD mRNA) within the positive picked samples from the
stimulated lungs. This matches well the increase in the target
mRNA/PBGD mRNA ratio of the lavaged AMs serving as reference. Two
aspects have to be addressed in further detail:
First, in the control lung, 17% of picked samples were positive for
TNF- mRNA. However, the TNF- mRNA expression in these positive
samples was stronger than in the lavaged AMs. This clearly suggests
that the analysis of (a large number of) lavaged cells averages the
expression of this cell population without considering individual
differences, whereas such individual differences between the cells are
revealed by the cell picking approach.
Second, concerning the quantitation of NOSII expression, the data
obtained from the picked AMs reflected closely those from the lavaged
AMs. A somewhat lower target gene mRNA/PBGD mRNA ratio was, however,
obtained for TNF- in picked AMs as compared to lavaged AMs from
stimulated lungs. This might be because of the fact that TNF- mRNA
is relatively unstable and short-lived, as compared to PBGD mRNA, which
may be assumed to be particularly relevant for the longer work-up
procedures for immunostaining at room temperature. In contrast, NOSII
mRNA is known to possess a long half-life (1 to 2 hours), which
moreover increases by LPS/IFN- stimulation.20,21
In
addition, nitric oxide is known to destabilize TNF- mRNA by reducing
its half-life.22
This type of reasoning again demonstrates
the necessity for an utmost short staining protocol.
In conclusion, when using an optimized protocol for immunofluorescence, laser-assisted cell picking can be effectively combined with real-time quantitative RT-PCR from a few immunodetected cells. The results obtained from lavaged AMs as reference confirmed the quantitative data obtained by cell picking. Thus, the combination with immunodetection is a substantial extension of the current technology allowing detection of pathophysiological gene regulation in a cell-specific manner within intact tissue structures.
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Acknowledgements |
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Footnotes |
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Supported by the Deutsche Forschungsgemeinschaft (SFB 547 "Cardiopulmonary vascular system," Project Z1).
Accepted for publication July 27, 2000.
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References |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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