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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.
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- MATERIALS AND METHODS
- 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.