close

Вход

Забыли?

вход по аккаунту

?

Quantitative Assessment of Macrophages in the Muscularis Externa of Mouse Intestines.

код для вставкиСкачать
THE ANATOMICAL RECORD 294:1557–1565 (2011)
Quantitative Assessment of Macrophages
in the Muscularis Externa of Mouse
Intestines
H.B. MIKKELSEN,1* J.O. LARSEN,2 P. FROH,1 AND T.H. NGUYEN1
Department of Cellular and Molecular Medicine, Faculty of Health Sciences,
University of Copenhagen, Denmark
2
Department of Neuroscience and Pharmacology, Faculty of Health Sciences,
University of Copenhagen, Denmark
1
ABSTRACT
Quantification of intestinal cells is challenging for several reasons:
The cell densities vary throughout the intestines and may be age dependent. Some cell types are ramified and/or can change shape and size. Additionally, immunolabeling is needed for the correct identification of cell
type. Immunolabeling is dependent on both up- and down-regulation of
the antigen being labeled as well as on the primary and secondary antibodies, the fixation, and the enhancement procedures. Here, we provide a
detailed description of immunolabeling of CD169þ cells and major histocompatibility class II antigen (MHCIIþ) cells and the subsequent quantification of these cells using design-based stereology in the intestinal
muscularis externa. We used young (5-weeks-old) and adult (10-weeksold) mice. Cell densities were higher in jejunum-ileum, when compared
with colon. In jejunum/ileum, the cell densities increased in oral-anal
direction in adults, whereas the densities were highest in the midpart in
young animals. In colon, the cell densities decreased in oral-anal direction
in both groups of animals. Except for the density of MHCIIþ cells in colon, the cell densities were highest in young animals. Densities of
CD169þ and MHCIIþ cells did not differ, except in the colon of young animals where the CD169þ density was almost twice as high as the MHCIIþ
density. CD169 and MHCII antigens seem to be expressed simultaneously
by the same cell in jejunum/ileum. We conclude that cell densities depend
on both the age of the mouse and on the location in the intestines. Anat
C 2011 Wiley-Liss, Inc.
Rec, 294:1557–1565, 2011. V
Key words: CD1691 cells; MHCII1 cells; macrophages; stereology;
mouse; intestine; young; adult
Robust quantitative data are often important in cell
characterization in experimental, developmental, and
pathologic studies. In intestinal motility disturbances,
for example, both the densities of interstitial cell of
Cajal, macrophages, and mast cells may be of interest
(Mikkelsen, 2010). Most studies of these cells are, however, based on semiquantitative techniques, where the
investigator counts cell profiles in few arbitrarily chosen
fields of visions in a specified region of the intestine or
measure the amount of fluorescence in sections. These
techniques for intestinal cell quantification are biased to
variable degrees. Both the number of cell profiles per
section area and the amount of fluorescence are functions of both the size and shape of the cells and of the
C 2011 WILEY-LISS, INC.
V
amount of intercellular tissue. As some of the cells are
ramified and can change their shape and/or size, it is
especially important to apply a counting rule that is
Grant sponsor: Vera and Carl Johan Michaelsens Foundation.
*Correspondence to: H.B. Mikkelsen, Department of Cellular
and Molecular Medicine, The Panum Building 22.4, Faculty of
Health Sciences, University of Copenhagen, Blegdamsvej 3,
2200 Copenhagen N, Denmark. E-mail: hannem@sund.ku.dk
Received 28 October 2010; Accepted 2 May 2011
DOI 10.1002/ar.21444
Published online 1 August 2011 in Wiley Online Library
(wileyonlinelibrary.com).
1558
MIKKELSEN ET AL.
number-weighted and not size- and/or shape-weighted.
In addition, the densities of intestinal cells vary regionally, which necessitates random sampling of fields of
visions.
Also in a recent study on gastrointestinal neuromuscular pathology, a need to standardize collection, processing, and quantification of neuronal and glial elements
in enteric neuropathologic samples was emphasized
(Knowles et al., 2009).
Here, we present a design-based stereological sampling technique that circumvents the sampling-related
problems associated with cell quantification. In order for
the actual application of this technique to be unbiased,
it is necessary to be able to correctly identify all the cells
of interest. Immunohistochemical methods are often
used to distinguish the cells at the light microscopic
level, and the applied staining techniques should be able
to either immunostain all cells of interest exclusively
without significant background staining or if more than
one cell type is being stained, the investigator should be
able to distinguish between them on the basis of location
and/or morphology or by using double staining. Mouse
macrophages can be labeled with several rat monoclonal
antibodies, but they possess the ability to change immunophenotype (and function) and the antibodies may not
stain the macrophage cell line exclusively. F4/80 antibody directed toward a plasma membrane glycoprotein
is the most commonly used macrophage marker but may
to some extent be less specific because it has also been
identified on cells of the following cell lines: monocytes,
eosinophils, and subgroups of dendritic cells (McGarry
and Stewart, 1991; Takahashi et al., 1992; Geissmann
et al., 2010). Additional drawbacks are that both special
fixation and enhancement techniques are recommended
to obtain an acceptable staining quality. Antibodies toward scavenger receptor class A (CD204) stain muscularis macrophages in outbred NMRI mice (Mikkelsen
et al., 2004), but because of a polymorphism of scavenger
receptor class A they are not usable in C57Bl/6 mice
(Daugherty et al., 2000). CD169 antibody, however, has
been recognized as a marker for metallophilic macrophages in the spleen, and has also been demonstrated to
be present on macrophages in the muscularis externa
(De Winter et al., 2005; Mikkelsen et al., 2008). The
macrophages in the small intestine express the major
histocompatibility class II antigens (MHCII) in conventionally housed adult mice, but not in newborn or germfree mice (Mikkelsen et al., 2004).
This study evaluates regional differences and age
related differences in the densities of MHCIIþ cells and
CD169þ cells with modern stereologic sampling. A
detailed description of the applied staining and quantification protocols are provided to acquaint potential users
in the field of intestinal motility to these tools for tissue
quantification. Double staining with MHCII and CD169
antibodies is not possible, partly because they require
different fixation protocols and partly because they both
are rat monoclonal antibodies. In the muscle layers, the
macrophages, that is, the F4/80þ, CD11bþ, and class A
scavenger receptorþ cells endocytose FITC-dextran
(Mikkelsen et al., 1988, 2004; Mikkelsen, 2010). As most
MHCIIþ cells also endocytose Fluoresceinisothiocyanatedextran (FITC-dextran) we presume them to represent the
same cell type. In this study we used FITC-dextran to
evaluate if the CD169þ cells (which possess an identical
morphology and distribution) have similar endocytic abilities and in this way represent the same cell type.
MATERIALS AND METHODS
Animals
Female specific pathogen-free C57Bl/6 (B6) mice (21)
were used (Taconic). We used six 5-weeks-old (young)
and six 10-weeks-old (adult) mice for the quantitative
main study, three animals for the FITC-dextran uptake
study, and additionally eight mice in a pilot study performed to examine for a potential differential shrinkage
following two different fixation procedures. The mice
were killed by cervical dislocation. All animals were
kept in a 12:12 light-dark cycle with free access to food
and water. All experimental procedures were in accordance with current national regulations issued by The
Danish Council on Animal Care.
Antibodies
The primary antibodies were rat anti-mouse MHC class
II antigen (Neomarkers A3-5, RT 946-P) (1:100) and rat
anti-CD169 (Serotec, MCA-884) (1:250). The secondary
antibodies were biotin conj. goat anti-rat (Amersham,
RPN 1005) (1:500), followed by StreptAB-complex/HRP
and DAB (DakoCytomation, using the recommendations
of the company or rhodamine-conjugated rabbit anti-rat
antibodies (Jackson) (1:100). Controls were incubated
with rat IgG2a (Serotec) and irrelevant rat antibodies.
Tissue Preparation and Immunohistochemistry
FITC-dextran uptake was examined in three animals
by injecting FITC dextran (MW 70.000, Sigma) 0.2 mL
0.71 mM in 154 mM NaCl intraperitoneally 24 hr before
sacrifice. In all animals, the intestines were removed to
prepare whole mount preparations, that is, stretched
preparations of muscularis externa, from jejunum-ileum
and colon. The removed part of jejunum-ileum started at
the first Peyer’s Patch and ended 1 cm before the ileocecal junction ( 26 cm long). The colon was taken from
the junction between cecum and proximal colon and as
distal as it was possibly to cut it (5–6 cm long). The
intestines were kept in Tris Buffered Saline on ice during the procedure, and the mucosa and submucosa were
removed with fine forceps and scissors under a stereomicroscope. The isolated muscle coats were placed in TBS
with nifedipine 1 lmol L1 to ensure relaxation, and the
muscle coats were pinned and stretched onto a Sylgard
plate. The same person performed the stretching and
pinning to avoid a potential bias arising from a differential stretching of the muscle coat. The muscle coat from
the colon was divided into four whole mounts. The muscle coat from jejunum-ileum was divided into approximately 14–16 whole mounts that were assigned
alternately (with a random start) to MHCII immunolabeling and CD169 immunolabeling, respectively. The
length of the whole mounts varied from 1.5 cm to 2 cm.
Whole mounts designated for MHCII immunolabeling
were fixed with 96% alcohol for 10 min and whole
mounts designated for CD169 immunolabeling were
fixed with 4% paraformaldehyde, pH: 7.4 for 3 hr. After
fixation, the pinned whole mounts were kept in TBS at
1559
CELL DENSITIES IN MOUSE INTESTINES
Fig. 1. Left: The counting rule for an isolated frame: Profiles completely or partly within the frame are counted provided that they do
not in any way touch any neighboring frames below or to the left of
the current frame, that is, profiles in the frame or at the (dashed) inclusion lines are counted, provided that they do not in any way touch the
(solid) exclusion lines or their infinite extensions. To apply this counting
rule, it is necessary to inspect an area around each counting frame,
that is, a guard area, to know the full extension of the profile. Three
profiles (drawn solid black) were counted in the counting frame. Right:
When a 2D-region is divided into rectangular frames (numbered consecutively from the lower left corner), a profile is counted the first time
it appears within a frame as one proceeds systematically through the
frames (drawn solid black in the frame where it is counted). The ability
to ensure correct definition of the profiles that belong to the area of
an isolated frame permits us to draw a sample of rectangles. If one
systematically inspects every second frame, that is, half the population, after randomly selecting the start within the first two frames,
50% of the time one will count in frames: 1, 3, 5, 7, and 9 (count 10
profiles), and 50% one will count in frames: 2, 4, 6, and 8 (count four
profiles). The mean of these two counts is 7, that is, the correct number of half the population. Inspired by Figure 1 in Larsen (1998).
4 C until immunostaining. All washing and incubation
solutions contained 0.5% triton-X 100. The tissue was
quenched in 1% H2O2 for 30 min and preincubated with
10% goat serum containing 0.5% Triton-X 100 to reduce
nonspecific staining. Primary and secondary antibodies
were diluted in TBS containing 0.5% Triton-X 100 and
10% goat serum. Incubations were done at 4 C; overnight for primary antibodies, 4 hr for biotin-conjugated
antibodies, and 2 hr for ABC-complex. The chromogen
was 0.5% diaminobenzidine in 0.035% H2O2. The whole
mounts were mounted with Aquatex (Merck). Rhodamine-conjugated antibodies were applied on whole
mounts from mice that had received FITC-dextran to do
double labeling.
A pilot study was conducted to test for a possible differential shrinkage of the whole mounts following the
two fixation procedures. The muscle coats through the
entire intestine were divided into whole mounts about
1.5 cm long. With a random start every second, whole
mount was fixed with 96% alcohol for 10 min and every
second with 4% paraformaldehyde, pH: 7.4 for 3 hr. The
whole mounts were measured after fixation and
mounting.
counted cells divided by the sum of counting frame
areas.
Stereological Analysis
The areal densities (i.e., the number of cells per surface area of the muscle coat), NA, of CD169þ and
MHCIIþ cells were estimated in the whole mounts from
the jejunum-ileum and colon. An unbiased counting
frame of area, a(frame), was positioned at coordinates of
a lattice of systematic, uniformly random points. The
number of cells, Q, within the counting frame was
counted through the full-thickness of the whole mount,
and the areal density was estimated as the sum of
P
NA :¼ P
Q
aðframeÞ
Densities were calculated both locally within the individual whole mount and globally for the entire jejunumileum and for the entire colon, respectively. The counting
rule and the sampling principle are shown in Fig. 1. The
stereological analysis was performed on a computer
monitor using computer-assisted interactive stereological
test systems (The CAST-grid software, Olympus, Denmark). Live video images of the fields of vision in the
microscope were transmitted by a video camera to the
computer screen. The microscope was equipped with
stepping motors that controlled stage movements via the
software. The entire region was delineated at a low magnification (102 using an 2 PlanApo objective). Cells
were counted at a final magnification of 1,024 using a
20 UPlanApo oil immersion objective (NA ¼ 0.8) to
which the 2 objective used for delineation was paracentered. At high magnification, the computer-controlled
stage of the Olympus BX51 microscope was programmed
to move the section systematically, random in a raster
pattern within the delineated region with interactively
defined steps separated by a distance of 600 lm in the xand y-axes, respectively. At each point in the raster
pattern, the image of an unbiased counting frame (of
area 5,232 lm2) was superimposed on to the microscope
image via the video-computer interface and was ‘‘moved’’
by moving the plane of focus through the entire thickness of the whole mount specimen, and all cells within
the unbiased counting frame were counted. The section
1560
MIKKELSEN ET AL.
Fig. 2. Immunostaining with CD169 antibody and MHCII antibody
in the muscularis of jejunum-ileum in young and adult animals. A, C,
E, and G express CD169. B, D, F, and H express MHCII. A, B, E, and
F are from young animals and C, D, G, and H are from adult animals.
A, B, C, and D are from proximal jejunum and E, F, G, and H are from
the distal ileum. Bar: 50 lm.
sampling fraction was 0.5, and the area sampling fraction was approximately 0.015.
In the pilot study, the length of each whole mount
preparation was measured using the CAST-grid ‘‘measure length’’ feature.
hyde, respectively, were calculated for each animal and
were compared using a two-tailed paired t-test. We
found no difference in the mean lengths (P ¼ 0.91) indicating that there was no differential shrinkage in alcohol
fixated gut and paraformaldehyde fixated gut in our protocols. Age related differences and the differences
between jejunum-ileum and colon were tested using a
two-way ANOVA, and the global densities of CD169þcells and MHCIIþ-cells were compared using a twotailed paired Student’s t-test.
STATISTICS
In the pilot study, the mean lengths of the whole
mounts fixated with 96% alcohol and 4% paraformalde-
CELL DENSITIES IN MOUSE INTESTINES
1561
Fig. 3. A gallery of confocal micrographs taken through the thickness of the jejunal muscularis in an adult mouse. A, B, and C are from
the serosa and D, E, and F are from the level of AP. G, H, and I are
from the level of the DMP. A and D show cells which have taken up
FITC-dextran and B and E are CD169þ cells. C and F show that the
cells which are CD169þ also have taken up FITC-dextran. At the level
of DMP, H and I show CD169þ oblong cells, but FITC-dextran uptake
is lacking in G and I. Bar: 30 lm.
RESULTS
Immunohistochemistry
Stereology
CD169þ cells had a morphology and distribution comparably with that of MHCIIþ cells along the intestine
both in young and adult mice (Fig. 2). In the small intestine, both the serosal cells and the cells at the level of
Auerbach’s plexus (AP) were ramified, and both cell
types contained small FITC-dextran vesicles. At the
level of the deep muscular plexus (DMP), a few oblong
CD169þ cells were observed. They did not contain FITCdextran (Fig. 3). In the colon, most serosal CD169þ and
MHCIIþ cells were oblong cells, but small cells without
ramifications were occasionally observed in scattered
groups. At AP, the cells were ramified, and in the circular muscular layer, an occasional bipolar cell was
observed (Fig. 4). In addition, we observed that young
mice had many round to oval cells in the proximal part
of colon (both CD169þ and MHCIIþ cells), whereas
in the distal part only few cells were scattered and the
densities were low.
Global cell densities are shown in Fig. 5, and the relevant statistical data when comparing young versus
adults and jejunum-ileum versus colon are given in Table 1. CD169þ cells versus MHCIIþ cells are compared
in Table 2. Young animals showed significantly higher
global densities of CD169þ cells in both jejunum-ileum
(35%) and in colon (31%) compared with adult mice (see
Table 1). As for MHCIIþ cells, the difference between
young and adult animals did not reach significance. The
group variances were nonsignificantly higher in adult
animals, when compared with young animals, except for
the group variances in the density of MHCIIþ cells in colon that was significantly higher in young animals (P ¼
0.03). For both cell types in both age groups, the cell
densities were significantly higher in jejunum-ileum
compared with colon. In young animals, the differences
were 16% and 57% for CD169þ cells and MHCIIþ cells,
respectively, and in adult animals, the corresponding differences were 13% and 28%. The global densities of
1562
MIKKELSEN ET AL.
Fig. 4. Immunostaining with CD169 antibody and MHCII antibody in the muscularis of colon in adult
animals. A and C express CD169 and B and D express MHCII. A and B are from the proximal part of colon, C and D are from the distal part. Bar: 50 lm.
in Fig. 5. In adult animals, both cell-types increased in
density in oral-anal direction in jejunum-ileum and
decreased in oral-anal direction in colon. In young animals, the decrease in oral-anal direction in colon is
equally clear, whereas it appears as if the densities in jejunum-ileum are higher in the midpart of jejunum-ileum. The first part of jejunum-ileum in adult mice
seems to have the smallest inter-animal variance in cell
densities (Fig. 6).
DISCUSSION
Fig. 5. The global cell densities of immunolabeled cells in each
mouse. Circles show young mice and squares show adult mice. Solid
symbols show the density of CD169þ-cells and open symbols the
density of MHCIIþ-cells. The horizontal lines give the group mean.
CD169þ-cells and MHCIIþ cells were apparently the
same in jejunum-ileum in both age groups and in colon
in the adult animals. In colon in young animals, there
was a significant higher density of CD169þ cells, when
compared with MHCIIþ cells (48% higher). The local cell
densities from each whole mount preparation are shown
Our study shows that the numbers of both MHCIIþ
and CD169þ cells vary along the intestinal tract. In
studies comparing cell densities, it is therefore optimal
to take several samples along the entire course of the
intestine or (if that is not possible as, e.g., in biopsy
studies) to at least select from the exact same region of
the intestine. Furthermore, unbiased counting and sampling principles should be applied to the sampled tissue.
‘‘Cell counts’’ based on counting cell profiles in few arbitrarily chosen fields of visions or based on measuring
the amount of fluorescence in sections are biased to variable degrees. The number of cell profiles per section
area depends on the size and shape of the cells as well
as on the amount of intercellular tissue as do the
amount of fluorescence emitted from a section. Recent
advances in automatic image analysis may when some
specific requirements are fulfilled provide reliable data.
Disadvantages are, that it cannot distinguish cells that
are part of a network and that one can only count in one
focal plane (and not through the entire section as we do)
1563
CELL DENSITIES IN MOUSE INTESTINES
TABLE 1. Comparing the cell densities in young animals versus adult animals (horizontal) and in
jejunum-ileum versus colon (vertical)
CD169þ-cells in jejunum-ileum
CD169þ-cells in colon
P-value
Difference
MHCIIþ-cells in jejunum-ileum
MHCIIþ-cells in colon
P-value
Difference
Young (n ¼ 6)
Adults (n ¼ 6)
P-value
Difference
271 (0.16)
228 (0.18)
0.0028
16%
279 (0.11)
119 (0.39)
0.00042
57%
201 (0.21)
174 (0.24)
0.00041
34.5%
30.6%
Ns
21.7%
28.2%
13%
229 (0.21)
166 (0.16)
28%
2
The table gives the group means (in number/mm ) for the global cell densities in jejunum-ileum and colon. The coefficient
of variation (SD/mean) is given in brackets. Horizontally, the cell densities of young animals (aged 5 weeks) are compared
with those of the adult animals (aged 10 weeks) and vertically the cell densities in jejunum-ileum are compared with those
in colon. The difference in group mean between young and adult animals is calculated as the difference between group
means divided by the group mean of the adults. The difference in group means of densities between jejunum-ileum and colon is calculated as the difference between the group means divided by the group mean of the density in jejunum-ileum.
TABLE 2. Comparing CD1691-cells versus MHCII1-cells (paired t-test)
CD169þ-cells
Jejunum-ileum in young animals (n ¼ 6)
Jejunum-ileum in adult animals (n ¼ 6)
Colon in young animals (n ¼ 6)
Colon in adult animals (n ¼ 6)
271
201
228
174
(0.16)
(0.21)
(0.18)
(0.24)
MHCIIþ-cells
279
229
119
166
(0.11)
(0.21)
(0.39)
(0.16)
P-value
Ns (0.710)
Ns (0.078)
0.0188
Ns (0.490)
Difference
48%
The table gives the group means of the cell densities of CD169þ-cells and MHCIIþ-cells (in number/mm2) in jejunum-ileum
and colon. The coefficient of variation (SD/mean) is given in brackets. The difference in group means is calculated as the
difference between the group means divided by the group mean of the density of CD169þ-cells.
so it is necessary that all cells are in focus in one plane.
Another drawback is that the fluorescence will fade in
time so that the sections cannot be stored too long before
the actual analysis take place. We have previously
shown that macrophages in mouse small intestine are
ramified in the serosa and at the level of AP, whereas a
more bipolar macrophage type resides in the circular
muscle layer (Mikkelsen et al., 1988; Mikkelsen, 1995,
2010). As the cells are ramified and can change their
shape and/or size, it is especially important to apply a
counting rule that is number-weighted and not sizeand/or shape-weighted. The counting can be performed
without the special equipment used in this study. It suffice to use a microscope with an unbiased counting
frame put into the eyepiece or alternatively a microscope, where the field of vision is video-transmitted to a
computer screen and superimposed with an unbiased
counting frame. The stage can be manually moved on
the stage-knob as the sampling only needs to be simple
random to be unbiased. The reason that it is an advantage to use systematic random sampling is that the precision of the estimate thus increases. One might approach
the systematic randomness by painting a mark on the
knob to approach uniform movements.
We found that the densities of CD169þ cells and
MHCIIþ cells were comparable in all regions of the
small intestine and that in adult mice cell densities
increased in oral-anal direction. In young animals, the
densities also differed in oral-anal direction but were in
that group highest in the mid-part of jejunum/ileum.
These findings suggest that CD169þ cells and MHCIIþ
cells represent the same macrophage subtype. However,
in a previous study on small intestinal macrophages, we
found that MHCIIþ cells outnumber F4/80þ cells (Mikkelsen et al., 2008) and therefore suggested the existence of (at least) two macrophage subtypes with similar
morphologies. An alternative explanation could be that
the macrophages express different activation state as
the F4/80 receptors seem to be down-regulated on macrophages in smooth muscle tissue and dense connective
tissue and up-regulated during alternative activation
(Mikkelsen, 2010). MHCII seem to be expressed by most
macrophages in conventionally housed mice (Mikkelsen
et al., 1988, 2004, 2008; Ozaki et al., 2004; Flores-Langarica et al., 2005; Bogunovic et al., 2009) but not by
macrophages in germ-free and newborn mice (Mikkelsen
et al., 2004). In recent years, nonlymphoid tissue dendritic cells have been described to be present in mice (Helft
et al., 2010). In the MHCII positive cell population of
mouse muscularis, presence of dendritic cells has been
described (Flores-Langarica et al., 2005). However, in a
recent study, only one cell population was found expressing a MHCIIhigh, CD11clow CD103, CD11bþ, F4/80þ
phenotype (Bogunovic et al., 2009). The cell population
was described to be derived from monocytes, had a
monocyte/macrophage morphology, and was responsive
to M-CSF. This is in accordance with our findings, where
we, in this study, have found that the CD169þ cells
located in serosa and at AP show FITC-dextran endocytosis and in previous studies have demonstrated that
FITC-dextran was endocytosed by all cells labeled with
antibodies toward F4/80, CD11b, and class A scavenger
receptor, and by most cells labeled with antibodies
toward MHCII (Mikkelsen et al., 1988, 2004).
We used the CD169 antibody as it appears to demonstrate most of the macrophages in muscularis externa of
1564
MIKKELSEN ET AL.
mouse intestine (Mikkelsen et al., 2008) and staining
with F4/80 and class A scavenger receptor antibodies differ in different mouse strains.
In colon of both adult and young animals, we found
lower densities of both CD169þ and MHCIIþ cells, when
compared with jejunum-ileum. Furthermore, in the colon
of the young animals, the density of CD169þ cells was
almost twice as high as that of MHCIIþ cells. Previously
studies on cell densities in the intestines are conflicting.
In a study on MHCIIþ cells in mouse small intestine
and colon, an increasing number of cells through small
intestine and colon was reported (Flores-Langarica
et al., 2005), whereas a study on ED-2þ macrophages in
rat intestines shows a significant higher density of macrophages in the small intestine, when compared with
the colon (Kalff et al., 1998), which is in accordance with
our findings in mouse colon. As MHCII is considered to
be up-regulated during classical activation and germfree and newborn mice are MHCII negative, this may
suggest less activation in colonic macrophages. It is surprising as the luminal content of the bacterial flora of
various regions (duodenum, jejunum, ileum, and large
intestine) of the gastrointestinal tracts differ (Mitsuoka,
2000) and bacteria in the small intestine is considered to
be less pathogenic than those of the colon (Marteau
et al., 2001). In jejunum-ileum, however, there is a
higher amount of antigens from ingested food, whereas
there are considerably less food antigens in colon. It has
also been shown that in the noninflamed intestinal mucosa, macrophages are noninflammatory but retain avid
scavenger and host defense functions (Smith et al.,
2005). The diversity in the number of the macrophages
in small intestine/colon may also reflect that small intestine is more active in motor function, which may cause
more damage and thereby repair processes. Altogether
this indicates that the variable MHCII expression may
reflect different activation states of the macrophages or
different subtypes of cells.
This study was done on a C57Bl/6 mouse strain; it is
probably the most widely used laboratory mouse strain,
due to the availability of congenic strains and easy
breeding. It is also the most widely used ‘‘genetic background’’ for genetically modified mice. However, the
results may differ in other mouse strains.
We can conclude that cell densities depend on both the
age of the mouse and on the location in the intestines.
The higher densities in young mice may be due to agerelated changes in the intestinal microflora pattern (Mitsuoka, 2000) or to a higher degree of tissue remodeling
as described to take place in embryonic and foetal mice
(Morris et al., 1991; Hopkinson-Woolley et al., 1994) and
the different densities in jejunum and colon may reflect
different microenvironments.
ACKNOWLEDGEMENTS
Fig. 6. The local cell densities in each mouse. Each graph is for an
individual animal where the x-axis is the section number (0 through 16
for jejunum-ileum and 0 through 4 for colon) and the y-axis the cell
density in that individual section. Circles show young mice, and
squares show adult mice. Solid symbols show the density of CD169þcells and open symbols the density of MHCIIþ-cells. The horizontal
lines give the group mean.
The authors thank Hanne Hadberg and Keld Ottesen
for skilled technical assistance.
LITERATURE CITED
Bogunovic M, Ginhoux F, Helft J, Shang L, Hashimoto D, Greter
M, Liu K, Jakubzick C, Ingersoll MA, Leboeuf M, Stanley ER,
Nussenzweig M, Lira SA, Randolph GJ, Merad M. 2009. Origin of
the lamina propria dendritic cell network. Immunity 31:513–525.
CELL DENSITIES IN MOUSE INTESTINES
Daugherty A, Whitman SC, Block AE, Rateri DL. 2000. Polymorphism of class A scavenger receptors in C57BL/6 mice. J Lipid
Res 41:1568–1577.
De Winter BY, Van Nassauw L, De Man JG, de Jonge F, Bredenoord
AJ, Seerden TC, Herman AG, Timmermans JP, Pelckmans PA.
2005. Role of oxidative stress in the pathogenesis of septic ileus
in mice. Neurogastroenterol Motil 17:251–261.
Flores-Langarica A, Meza-Perez S, Calderon-Amador J, EstradaGarcia T, Macpherson G, Lebecque S, Saeland S, Steinman RM,
Flores-Romo L. 2005. Network of dendritic cells within the muscular layer of the mouse intestine. Proc Natl Acad Sci USA 102:
19039–19044.
Geissmann F, Gordon S, Hume DA, Mowat AM, Randolph GJ. 2010.
Unravelling mononuclear phagocyte heterogeneity. Nat Rev
Immunol 10:453–460.
Helft J, Ginhoux F, Bogunovic M, Merad M. 2010. Origin and functional heterogeneity of non-lymphoid tissue dendritic cells in
mice. Immunol Rev 234:55–75.
Hopkinson-Woolley J, Hughes D, Gordon S, Martin P. 1994. Macrophage recruitment during limb development and wound healing
in the embryonic and foetal mouse. J Cell Sci 107(Pt 5):1159–
1167.
Kalff JC, Schwarz NT, Walgenbach KJ, Schraut WH, Bauer AJ.
1998. Leukocytes of the intestinal muscularis: their phenotype
and isolation. J Leukoc Biol 63:683–691.
Knowles CH, De Giorgio R, Kapur RP, Bruder E, Farrugia G,
Geboes K, Gershon MD, Hutson J, Lindberg G, Martin JE, MeierRuge WA, Milla PJ, Smith VV, Vandervinden JM, Veress B, Wedel
T. 2009. Gastrointestinal neuromuscular pathology: guidelines for
histological techniques and reporting on behalf of the Gastro 2009
International Working Group. Acta Neuropathol 118:271–301.
Larsen JO. 1998. Stereology of nerve cross sections. J Neurosci
Methods 85:107–118.
Marteau P, Pochart P, Dore J, Bera-Maillet C, Bernalier A, Corthier
G. 2001. Comparative study of bacterial groups within the human
cecal and fecal microbiota. Appl Environ Microbiol 67: 4939–
4942.
1565
McGarry MP, Stewart CC. 1991. Murine eosinophil granulocytes
bind the murine macrophage-monocyte specific monoclonal antibody F4/80. J Leukoc Biol 50:471–478.
Mikkelsen HB. 1995. Macrophages in the external muscle layers of
mammalian intestines. Histol Histopathol 10:719–736.
Mikkelsen HB. 2010. Interstitial cells of Cajal, macrophages and
mast cells in the gut musculature: morphology, distribution, spatial and possible functional interactions. J Cell Mol Med 14:818–
832.
Mikkelsen HB, Garbarsch C, Tranum-Jensen J, Thuneberg L. 2004.
Macrophages in the small intestinal muscularis externa of embryos,
newborn and adult germ-free mice. J Mol Histol 35:377–387.
Mikkelsen HB, Larsen JO, Hadberg H. 2008. The macrophage system in the intestinal muscularis externa during inflammation: an
immunohistochemical and quantitative study of osteopetrotic mice.
Histochem Cell Biol 130:363–373.
Mikkelsen HB, Mirsky R, Jessen KR, Thuneberg L. 1988. Macrophage-like cells in muscularis externa of mouse small intestine:
Immunohistochemical localization of F4/80, M1/70, and Ia-antigen. Cell Tissue Res 252:301–306.
Mitsuoka T. 2000. Significance of dietary modulation of intestinal
flora and intestinal environment. Biosci Microflora 19:15–25.
Morris L, Graham CF, Gordon S. 1991. Macrophages in haemopoietic and other tissues of the developing mouse detected by the
monoclonal antibody F4/80. Development 112:517–526.
Ozaki H, Kawai T, Shuttleworth CW, Won KJ, Suzuki T, Sato K,
Horiguchi H, Hori M, Karaki H, Torihashi S, Ward SM, Sanders
KM. 2004. Isolation and characterization of resident macrophages
from the smooth muscle layers of murine small intestine. Neurogastroenterol Motil 16:39–51.
Smith PD, Ochsenbauer-Jambor C, Smythies LE. 2005. Intestinal
macrophages: unique effector cells of the innate immune system.
Immunol Rev 206:149–159.
Takahashi K, Naito M, Schultz LD. 1992. Differentiation of epidermal Langerhans cells in macrophage colony-stimulating-factor-deficient mice homozygous for the osteopetrosis (op) mutation. J
Invest Dermatol 99:46S–47S.
Документ
Категория
Без категории
Просмотров
2
Размер файла
1 852 Кб
Теги
macrophage, intestinal, mouse, external, assessment, muscular, quantitative
1/--страниц
Пожаловаться на содержимое документа