SEARCH

SEARCH BY CITATION

Keywords:

  • CD169+ cells;
  • MHCII+ cells;
  • macrophages;
  • stereology;
  • mouse;
  • intestine;
  • young;
  • adult

Abstract

  1. Top of page
  2. Abstract
  3. MATERIALS AND METHODS
  4. STATISTICS
  5. RESULTS
  6. DISCUSSION
  7. Acknowledgements
  8. LITERATURE CITED

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 CD169+ cells and major histocompatibility class II antigen (MHCII+) 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-weeks-old) 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 MHCII+ cells in colon, the cell densities were highest in young animals. Densities of CD169+ and MHCII+ cells did not differ, except in the colon of young animals where the CD169+ density was almost twice as high as the MHCII+ 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 Rec, 2011. © 2011 Wiley-Liss, Inc.

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 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 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 MHCII+ cells and CD169+ 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 F4/80+, CD11b+, and class A scavenger receptor+ cells endocytose FITC-dextran (Mikkelsen et al.,1988,2004; Mikkelsen,2010). As most MHCII+ cells also endocytose Fluoresceinisothiocyanate-dextran (FITC-dextran) we presume them to represent the same cell type. In this study we used FITC-dextran to evaluate if the CD169+ cells (which possess an identical morphology and distribution) have similar endocytic abilities and in this way represent the same cell type.

MATERIALS AND METHODS

  1. Top of page
  2. Abstract
  3. MATERIALS AND METHODS
  4. STATISTICS
  5. RESULTS
  6. DISCUSSION
  7. Acknowledgements
  8. LITERATURE CITED

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 μmol L−1 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 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.

Stereological Analysis

The areal densities (i.e., the number of cells per surface area of the muscle coat), NA, of CD169+ and MHCII+ 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 counted cells divided by the sum of counting frame areas.

  • equation image

Densities were calculated both locally within the individual whole mount and globally for the entire jejunum-ileum 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 = 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 μm in the x- and y-axes, respectively. At each point in the raster pattern, the image of an unbiased counting frame (of area 5,232 μm2) 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 sampling fraction was 0.5, and the area sampling fraction was approximately 0.015.

thumbnail image

Figure 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).

Download figure to PowerPoint

In the pilot study, the length of each whole mount preparation was measured using the CAST-grid “measure length” feature.

STATISTICS

  1. Top of page
  2. Abstract
  3. MATERIALS AND METHODS
  4. STATISTICS
  5. RESULTS
  6. DISCUSSION
  7. Acknowledgements
  8. LITERATURE CITED

In the pilot study, the mean lengths of the whole mounts fixated with 96% alcohol and 4% paraformaldehyde, 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 = 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 CD169+-cells and MHCII+-cells were compared using a two-tailed paired Student's t-test.

RESULTS

  1. Top of page
  2. Abstract
  3. MATERIALS AND METHODS
  4. STATISTICS
  5. RESULTS
  6. DISCUSSION
  7. Acknowledgements
  8. LITERATURE CITED

Immunohistochemistry

CD169+ cells had a morphology and distribution comparably with that of MHCII+ 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 CD169+ cells were observed. They did not contain FITC-dextran (Fig. 3). In the colon, most serosal CD169+ and MHCII+ 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 CD169+ and MHCII+ cells), whereas in the distal part only few cells were scattered and the densities were low.

thumbnail image

Figure 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 μm.

Download figure to PowerPoint

thumbnail image

Figure 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 CD169+ cells. C and F show that the cells which are CD169+ also have taken up FITC-dextran. At the level of DMP, H and I show CD169+ oblong cells, but FITC-dextran uptake is lacking in G and I. Bar: 30 μm.

Download figure to PowerPoint

thumbnail image

Figure 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 μm.

Download figure to PowerPoint

Stereology

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. CD169+ cells versus MHCII+ cells are compared in Table 2. Young animals showed significantly higher global densities of CD169+ cells in both jejunum-ileum (35%) and in colon (31%) compared with adult mice (see Table 1). As for MHCII+ 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 MHCII+ cells in colon that was significantly higher in young animals (P = 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 CD169+ cells and MHCII+ cells, respectively, and in adult animals, the corresponding differences were 13% and 28%. The global densities of CD169+-cells and MHCII+ 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 CD169+ cells, when compared with MHCII+ cells (48% higher). The local cell densities from each whole mount preparation are shown 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).

thumbnail image

Figure 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 CD169+-cells and open symbols the density of MHCII+-cells. The horizontal lines give the group mean.

Download figure to PowerPoint

thumbnail image

Figure 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 CD169+-cells and open symbols the density of MHCII+-cells. The horizontal lines give the group mean.

Download figure to PowerPoint

Table 1. Comparing the cell densities in young animals versus adult animals (horizontal) and in jejunum-ileum versus colon (vertical)
 Young (n = 6)Adults (n = 6)P-valueDifference
  1. The table gives the group means (in number/mm2) 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.

CD169+-cells in jejunum-ileum271 (0.16)201 (0.21)0.0004134.5%
CD169+-cells in colon228 (0.18)174 (0.24)30.6%
P-value0.0028 
Difference16%13%
MHCII+-cells in jejunum-ileum279 (0.11)229 (0.21)Ns21.7%
MHCII+-cells in colon119 (0.39)166 (0.16)28.2%
P-value0.00042 
Difference57%28%
Table 2. Comparing CD169+-cells versus MHCII+-cells (paired t-test)
 CD169+-cellsMHCII+-cellsP-valueDifference
  1. The table gives the group means of the cell densities of CD169+-cells and MHCII+-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 CD169+-cells.

Jejunum-ileum in young animals (n = 6)271 (0.16)279 (0.11)Ns (0.710) 
Jejunum-ileum in adult animals (n = 6)201 (0.21)229 (0.21)Ns (0.078) 
Colon in young animals (n = 6)228 (0.18)119 (0.39)0.018848%
Colon in adult animals (n = 6)174 (0.24)166 (0.16)Ns (0.490) 

DISCUSSION

  1. Top of page
  2. Abstract
  3. MATERIALS AND METHODS
  4. STATISTICS
  5. RESULTS
  6. DISCUSSION
  7. Acknowledgements
  8. LITERATURE CITED

Our study shows that the numbers of both MHCII+ and CD169+ 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) 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 size- and/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 CD169+ cells and MHCII+ 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 CD169+ cells and MHCII+ cells represent the same macrophage subtype. However, in a previous study on small intestinal macrophages, we found that MHCII+ cells outnumber F4/80+ 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÷, CD11b+, F4/80+ 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 CD169+ 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 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 CD169+ and MHCII+ cells, when compared with jejunum-ileum. Furthermore, in the colon of the young animals, the density of CD169+ cells was almost twice as high as that of MHCII+ cells. Previously studies on cell densities in the intestines are conflicting. In a study on MHCII+ 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-2+ 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 germ-free 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 age-related 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

  1. Top of page
  2. Abstract
  3. MATERIALS AND METHODS
  4. STATISTICS
  5. RESULTS
  6. DISCUSSION
  7. Acknowledgements
  8. LITERATURE CITED

The authors thank Hanne Hadberg and Keld Ottesen for skilled technical assistance.

LITERATURE CITED

  1. Top of page
  2. Abstract
  3. MATERIALS AND METHODS
  4. STATISTICS
  5. RESULTS
  6. DISCUSSION
  7. Acknowledgements
  8. LITERATURE CITED