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Macrophages in the Small Intestinal Muscularis Externa of Embryos, Newborn and Adult Germ-Free Mice

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Abstract

Previously, we demonstrated the presence of a constant and regularly distributed macrophage population of ramified cells in the intestinal muscle layers of smaller rodents. The function of these resident macrophages under normal conditions remains unknown. Histochemistry, immunohistochemistry and electron microscopy were applied to the muscularis externa of 15- and 17-day-old embryos, 2-day-old mice, adult germ-free and conventional mice. Since lipopolysaccharides (LPS) activates macrophages and inflammation affects gut motility, LPS-treated mice were also included in the study. Two macrophage antibodies, F4/80 and 2F8 were used to demonstrate the presence of macrophages in the muscle layers. The localization was confirmed by electron microscopy. In contrast to conventional adult mice, the muscle layers in embryos, newborn and germ-free adult mice were devoid of class II MHC antigen reactive cells. The acid phosphatase reaction and antibodies directed towards a lysosomal protein (Lamp-2) were used in order to verify other activation markers. None of these showed specific staining of the muscularis macrophages. Only LPS-treated adult mice showed iNOS-positive cells in whole mounts. We conclude that the characteristic organization and distribution of muscularis macrophages in adult mice are also present in embryos, newborn and germ-free mice and thus develop independently of foreign antigens. Further, these macrophages are truly resident and appear to have differential responses to exogene stimuli.

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References

  • Anderson CR, Edwards SL (1993) Subunit b of cholera toxin labels interstitial cells of Cajal in the gut of rat and mouse. Histochemistry 100: 457–464.

    Google Scholar 

  • Austyn JM, Gordon S (1981) F4/80, a monoclonal antibody directed specifically against the mouse macrophage. Eur J Immunol 11: 805–815.

    Google Scholar 

  • Eskandari MK, Kalff JC, Billiar TR, Lee KKW, Bauer AJ (1997) Lipopolysaccharide activates the muscularis macrophage network and suppresses circular smooth muscle activity. Am J Physiol 273: G727–G734.

    Google Scholar 

  • Eskandari MK, Kalff JC, Billiar TR, Lee KKW, Bauer AJ (1999) LPS-induced muscularis macrophage nitric oxide suppresses jejunal circular muscle activity. Am J Physiol 277: G478–G486.

    Google Scholar 

  • Faussone-Pellegrini MS (1985) Cytodifferentiation of the interstitial cells of Cajal related to the myenteric plexus of mouse intestinal muscle coat. An E.M. study from foetal to adult life. Anat Embryol 171: 163–169.

    Google Scholar 

  • Galeazzi F, Haapala EM, van Rooijen N, Vallance BA, Collins SM (2000) Inflammation-induced impairment of enteric nerve function in nematode-infected mice is macrophage dependent. Am J Physiol 278: G259–G265.

    Google Scholar 

  • Gordon S, Lawson L, Rabinowitz S, Crocker PR, Morris L, Perry VH (1992) Antigen markers of macrophage differentiation in murine tissues. In: Gordon S, ed. Macrophage Biology and Activation (Current Topics in Microbiology and Immunology) Vol. 181. Berlin, Heidelberg, New York: Springer, pp. 1–37.

    Google Scholar 

  • Hoffman RA, Zhang G, Nussler NC, Gleixner SL, Ford HR, Simmons RL, Watkins SC (1997) Constitutive expression of inducible nitric oxide synthase in the mouse ileal mucosa. Am J Physiol 272: G383–G392.

    Google Scholar 

  • Hori M, Kita M, Torihashi S, Miyamoto S, Won KJ, Sato K, Ozaki H, Karaki H (2001) Upregulation of iNOS by COX-2 in muscularis resident macrophage of rat intestine stimulated with LPS. Am J Physiol Gastrointest Liver Physiol 280: G930–G938.

    Google Scholar 

  • Hughes DA, Gordon S (1998) Expression and function of the type 3 complement receptor in tissues of the developing mouse. J Immunol 160: 4543–4552.

    PubMed  Google Scholar 

  • Huizinga JD, Thuneberg L, Klüppel M, Malysz J, Mikkelsen HB, Bernstein A (1995) W/kitgene required for interstitial cells of Cajal and for intestinal pacemaker activity. Nature 373: 347–349.

    Google Scholar 

  • Hume DA, Monkley SJ, Wainright B (1995) Detection of c-fmsprotooncogene in early mouse embryos by whole mount in situhybridization indicates roles for macrophage in tissue remodelling. Br J Haematol 90: 939–942.

    Google Scholar 

  • 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.

    Google Scholar 

  • Kennedy DW, Abkowitz JL (1998) Mature monocytic cells enter tissues and engraft. Proc Natl Acad Sci USA 95: 14944–14949.

    Google Scholar 

  • Liu LWC, Thuneberg L, Huizinga JD (1998) Development of pacemaker activity and interstitial cells of Cajal in the neonatal mouse small intestine. Dev Dynam 213: 271–282.

    Google Scholar 

  • McKnight AJ, Gordon S (1998) Membrane molecules as differentiation antigens of murine macrophages. In: Dixon FJ, ed. Advances in Immunology, Vol. 68. San Diego, London, Boston: Academic Press, pp. 271–414.

    Google Scholar 

  • Meijer AE (1972) Semipermeable membranes for improving the histochemical demonstration of enzyme activities in tissue sections. I. Acid phosphatase. Histochemie 30: 31–39.

    Google Scholar 

  • Mikkelsen HB (1995) Macrophages in the external muscle layers of mammalian intestines. Histol Histopathol 10: 719–736.

    PubMed  Google Scholar 

  • Mikkelsen HB, Malysz J, Huizinga JD, Thuneberg L (1998) Action potential generation, Kit receptor immunohistochemistry and morphology of steel-Dickie (Sl/Sld )mutant mouse small intestine. Neurogastroenterol Mot 10: 11–26.

    Google Scholar 

  • Mikkelsen HB, MirskyR, Jessen KR, Thuneberg L (1988a) Macrophagelike cells in muscularis externa of mouse small intestine: Immunohistochemical localization of F4/80, M1/70, and Ia-antigen. Cell Tissue Res 252: 301–306.

    Google Scholar 

  • Mikkelsen HB, Rumessen JJ (1992) Characterization of macrophagelike cells in the external layers of human small and large intestine. Cell Tissue Res 270: 273–279.

    Google Scholar 

  • Mikkelsen HB, Thuneberg L (1999) Op/opmice defective in production of functional colony-stimulating factor-1 lack macrophages in muscularis externa of the small intestine. Cell Tissue Res 295: 485–493.

    PubMed  Google Scholar 

  • Mikkelsen HB, Thuneberg L, Rumessen JJ, Thorball N (1985) Macrophage-like cells in the muscularis externa of mouse small intestine. Anat Rec 213: 77–86.

    PubMed  Google Scholar 

  • Mikkelsen HB, Thuneberg L, Wittrup IH (1988b) Selective double staining of interstitial cells of Cajal and macrophage-like cells in small intestine by an improved supravital methylene blue technique combined with FITC-dextran uptake. Anat Embryol 178: 191–195.

    Google Scholar 

  • 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.

    Google Scholar 

  • Qu XW, Rozenfeld RA, Huang W, Sun X, Tan XD, Hsueh W (1999) Roles of nitric oxide synthases in platelet-activating factor-induced intestinal necrosis in rats. Crit Care Med 27: 356–364.

    PubMed  Google Scholar 

  • Rühl A, Berezin I, Collins SM (1995) Involvement of eicosanoids and macrophage-like cells in cytokine-mediated changes in rat myenteric nerves. Gastroenterology 109: 1852–1862.

    PubMed  Google Scholar 

  • Sadlack B, Merz H, Schorle H, Schimpl A, Feller AC, Horak I (1993) Ulcerative colitis-like disease in mice with a disrupted interleukin-2 gene. Cell 75: 253–261.

    PubMed  Google Scholar 

  • Schultz M, Tonkonogy SL, Sellon RK, Veltkamp C, Godfrey VL, Kwon J, Grenther WB, Balish E, Horak I, Sartor RB (1999) IL-2-deficient mice raised under germfree conditions develop delayed mild focal intestinal inflammation. Am J Physiol 276: G1461–G1472.

    PubMed  Google Scholar 

  • Sellon RK, Tonkonogy S, Schultz M, Dieleman LA, Grenther W, Balish E, Rennick DM, Sartor RB (1998) Resident enteric bacteria are necessary for development of spontaneous colitis and immune system activation in interleukin-10-deficient mice. Infect Immun 66: 5224–5231.

    Google Scholar 

  • Stoward PJ, Al Sarraj B (1981) Quantitative histochemical investigations of semipermeable membrane techniques for the assay of acid phosphatase in skeletal muscle. I. Meijer's method. Histochemistry 71: 405–431.

    Google Scholar 

  • Takahashi K, Donovan MJ, Rogers RA, Ezekowitz RAB(1998) Distribution of murine mannose receptor expression from early embryogenesis through adulthood. Cell Tissue Res 292: 311–323.

    Google Scholar 

  • Taurog JD, Richardson JA, Croft JT, Simmons WA, Zhou M, Fernandez-Sueiro JL, Balish E, Hammer RE (1994) The germfree state prevents development of gut and joint inflammatory disease in HLA-B27 transgenic rats. J Exp Med 180: 2359–2364.

    Article  PubMed  Google Scholar 

  • Thuneberg L (1982) Interstitial cells of Cajal: Intestinal pacemaker cells? Adv Anat Embryol Cell Biol 71: 1–130.

    PubMed  Google Scholar 

  • Torihashi S, Ozaki H, Hori M, Kita M, Ohota S, KarakiH(2000) Resident macrophages activated by lipopolysaccharide suppress muscle tension and initiate inflammatory response in the gastrointestinal muscle layer. Histochem Cell Biol 113: 73–80.

    Article  Google Scholar 

  • Torihashi S, Ward SM, Nishikawa SI, Nishi K, Kobayashi S, Sanders KM (1995) c-kit-dependent development of interstitial cells and electrical activity in the murine gastrointestinal tract. Cell Tissue Res 280: 97–111.

    Google Scholar 

  • Torihashi S, Ward SM, Sanders KM(1997) Development of c-kit-positive cells and the onset of electrical rhythmicity in murine small intestine. Gastroenterology 112: 144–155.

    Google Scholar 

  • Umesaki Y, Setoyama H (2000) Structure of the intestinal flora responsible for development of the gut immune system in a rodent model. Microbes Infect 2: 1343–1351.

    Google Scholar 

  • Wang XY, Berezin I, Mikkelsen HB, Der T, Bercik P, Collins SM, Huizinga JD (2002) Pathology of interstitial cells of Cajal in relation to inflammation revealed by ultrastructure but not immunohistochemistry. Am J Pathol 160: 1529–1540.

    Google Scholar 

  • Young HM, Mc Conalogue K, Furness JB, DeVente J (1993) Nitric oxide targets in the guinea-pig intestine identified by induction of cyclicGMP immunoreactivity. Neuroscience 55: 583–596.

    Google Scholar 

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Mikkelsen, H.B., Garbarsch, C., Tranum-Jensen, J. et al. Macrophages in the Small Intestinal Muscularis Externa of Embryos, Newborn and Adult Germ-Free Mice. Histochem J 35, 377–387 (2004). https://doi.org/10.1023/B:HIJO.0000039840.86420.b7

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  • DOI: https://doi.org/10.1023/B:HIJO.0000039840.86420.b7

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