Quantitative assessment of macrophages in the muscularis externa of mouse intestines.

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.

Bacterial adherence in otitis media: determination of N-acetylgalactosamine (GalNAc) residues in the submucosal glands and surface epithelium of the normal and diseased Eustachian tube.

BACKGROUND: Acute otitis media (AOM) is the most common childhood infection caused by bacteria. The pathogenesis of AOM implicates initial adherence of a pathogen to the nasopharyngeal epithelium, which is followed by bacterial colonization of the middle ear cavity through the Eustachian tube. N-acetylgalactosamine (GalNAc) is an important constituent of mucins and GalNAc containing sugar residues seem to be essential for initial adherence of respiratory bacteria to the surface of epithelial cells. OBJECTIVE: To explore the localization of GalNAc residues, we incubated Eustachian tube sections from Streptococcus pneumoniae infected and normal control rats with seven biotinylated, GalNAc recognizing lectins: Bauhinia purpurea lectin (BPA), Psophocarpus tetragonolobus lectin (PTA), Helix aspersa lectin (HAA), Helix pomatia lectin (HPA), Phaseolus lunatus lectin (PLA), Sophora japonica lectin (SJA) and Vicia Villosa isolectin B4 (VVA-B4). RESULTS: The mucin producing epithelium and submucosal glands of the normal Eustachian tube contained GalNAc residues, as evidenced by binding of several of the lectins. Lectin binding specificity and intensity changed following acute middle ear infection. BPA was the only lectin that exclusively stained the surface epithelium and the serous acini of the submucosal glands in the infected animals, whereas no binding was detected in the normal controls. HPA, HAA, PTA and VVA-B4 binding to surface epithelial cells increased after infection, indicating an active secretion of GalNAc containing glycans. Quantitative analysis of submucosal gland staining intensity showed significantly more GalNAc residues in the normal Eustachian tube, compared to infected animals. CONCLUSION: We conclude that the mucous producing elements of the normal rat Eustachian tube contain GalNAc residues essential for respiratory pathogen adherence. In addition, the GalNAc residue specificity and reacting intensity change in relation to acute infection, which may be important in relation to subsequent development of secretory otitis media or formation of a bacterial biofilm in the middle ear. The results show that GalNAc residues increased in both the submucosal serous glands and in the surface epithelium of the Eustachian tube after middle ear infection with S. pneumoniae.

Igf1r+/CD34+ immature ICC are putative adult progenitor cells, identified ultrastructurally as fibroblast-like ICC in Ws/Ws rat colon.

The colon of Ws/Ws mutant rats shows impairment of pacemaker activity and altered inhibitory neurotransmission. The present study set out to find structural correlates to these findings to resolve mechanisms. In the colon of Ws/Ws rats, interstitial cells of Cajal associated with Auerbach's plexus (ICC-AP) were significantly decreased and ICC located at the submuscular plexus and intramuscular ICC were rarely observed based on immunohistochemistry and electron microscopy. Ultrastructural investigations revealed that there was no overall loss of all types of interstitial cells combined. Where loss of ICC was observed, a marked increase in fibroblast-like ICC (FL-ICC) was found at the level of AP. Immunoelectron microscopy proved FL-ICC to be c-Kit(-) but gap junction coupled to each other and to c-Kit(+) ICC; they were associated with enteric nerves and occupied space normally occupied by ICC in the wild-type rat colon, suggesting them to be immature ICC. In addition, a marked increase in immunoreactivity for insulin-like growth factor 1 receptor (Igf1r) occurred, co-localized with CD34 but not with c-Kit. A significantly higher number of Igf1r(+)/CD34(+) cells were found in Ws/Ws compared to wild-type rat colons. These CD34(+)/Igf1r(+) cells in the Ws/Ws colon occupied the same space as FL-ICC. Hence we propose that a subset of immature ICC (FL-ICC) consists of adult progenitor cells. Immunohistochemistry revealed a reduction of neurons positive for neuronal nitric oxide synthase. The functional capabilities of the immature ICC and the regenerative capabilities of the adult progenitor cells need further study. The morphological features described here show that the loss of pacemaker activity is not associated with failure to develop a network of interstitial cells around AP but a failure to develop this network into fully functional pacemaker cells. The reduction in nitrergic innervation associated with the Ws mutation may be the result of a reduction in nitrergic neurons.

The macrophage system in the intestinal muscularis externa during inflammation: an immunohistochemical and quantitative study of osteopetrotic mice.

Intestinal inflammation results in disturbed intestinal motility in humans as well as in animal models. This altered function of smooth muscle cells and/or the enteric nervous system may be caused by activation of macrophages in muscularis externa and a thereby following release of cytokines and chemokines that causes influx of mononuclear cells and neutrophilic granulocytes. We subjected osteopetrotic (op/op) mice that lack certain macrophage subtypes, e.g. macrophages in the muscularis externa and +/+ mice to LPS to induce inflammatory cell influx. The densities of F4/80+, MHCII+, and myeloperoxidase+ cells were quantified using stereological sampling. In +/+ mice we found that MHCII+ cells outnumber F4/80+ cells and that LPS injection increased the density of MHCII+ cells temporarily but not that of F4/80+ cells. This indicates that an upregulation of MHCII antigen takes place and that two or more macrophage subtypes with comparable morphologies exist. Osteopetrotic mice lacked MHCII+, CD169+, and F4/80+ cells after either treatment, which indicate that these cells are CSF-1-dependent. LPS induced VCAM-1 activation of the vessels, modest influx of granulocytes, as well as an iNOS-activation in a cell type different from macrophages in both +/+ and op/op mice.

Pacemaker activity and inhibitory neurotransmission in the colon of Ws/Ws mutant rats.

The aim of this study was to characterize the pacemaker activity and inhibitory neurotransmission in the colon of Ws/Ws mutant rats, which harbor a mutation in the c-kit gene that affects development of interstitial cells of Cajal (ICC). In Ws/Ws rats, the density of KIT-positive cells was markedly reduced. Wild-type, but not Ws/Ws, rats showed low- and high-frequency cyclic depolarization that were associated with highly regular myogenic motor patterns at the same frequencies. In Ws/Ws rats, irregular patterns of action potentials triggered irregular muscle contractions occurring within a bandwidth of 10-20 cycles/min. Spontaneous activity of nitrergic nerves caused sustained inhibition of muscle activity in both wild-type (+/+) and Ws/Ws rats. Electrical field stimulation of enteric nerves, after blockade of cholinergic and adrenergic activity, elicited inhibition of mechanical activity and biphasic inhibitory junction potentials both in wild-type and Ws/Ws rats. Apamin-sensitive, likely purinergic, inhibitory innervation was not affected by loss of ICC. Variable presence of nitrergic innervation likely reflects the presence of direct nitrergic innervation to smooth muscle cells as well as indirect innervation via ICC. In summary, loss of ICC markedly affects pacemaker and motor activities of the rat colon. Inhibitory innervation is largely maintained but nitrergic innervation is reduced possibly related to the loss of ICC-mediated relaxation.

Motility patterns and distribution of interstitial cells of Cajal and nitrergic neurons in the proximal, mid- and distal-colon of the rat.

The aim of this work was to study the patterns of spontaneous motility in the circular and longitudinal muscle strips and to characterize the distribution of c-kit positive interstitial cells of Cajal (ICCs) and nitrergic neurons (nNOS) in the proximal, mid- and distal-colon of Sprague-Dawley rats. We described two types of spontaneous contractions: high frequency (HF) and low frequency (LF) contractions, which were recorded in the presence of tetrodotoxin, suggesting a non-neurogenic origin. Regional differences were found in the motility patterns depending on the muscle layer and on the part of the colon studied. Muscle strips without submuscular plexus (SMP) showed only LF contractions. The density of ICCs was of the same magnitude along the extent of the colon: about 90-120 cells mm(-2) at Auerbach's plexus (AP) and 50-60 cells mm(-2) at the SMP. nNOS positive cells were found at the level of the AP and the major density was found in the mid-colon. Electrical field stimulation abolished LF but did not affect HF contractions. Our results indicate that HF contractions are due to the ICC network found associated with the submuscular plexus (ICC-SMP). The origin of LF contractions is still unknown.

Expression of connexin 37, 40, and 43 mRNA and protein in renal preglomerular arterioles.

Gap junctions allow direct intercellular coupling between many cells including those in the vascular wall. Studies of connexin expression in cells of the microcirculatory system are very few in number. However, cell-to-cell communication between cells of the arteriolar wall may be particularly important in microcirculatory control. We investigated the expression of connexins 43, 40, and 37 (Cx43, Cx40, Cx37) mRNA and proteins in primary cultures of smooth muscle cells (SMC) from rat renal preglomerular arterioles and in the aortic cell line A7r5. Furthermore protein expression in preglomerular arterioles in frozen sections was evaluated. SMC were isolated from kidneys using an iron oxide sieve method and explant technique. Total RNA from these cultures was tested by RT-PCR analysis for the expression of the three connexins mRNA. Using immunofluorescence we examined whether the expression pattern of connexin protein in the cell culture and frozen sections corresponded to the mRNA expression. The data show that A7r5 and preglomerular SMC express mRNA for Cx37 in addition to Cx43 and Cx40. In A7r5 cells the mRNA for Cx43, Cx40, and Cx37 are translated to protein, whereas cultured preglomerular SMC and the media of afferent arterioles in frozen sections only showed Cx40 immunoreactivity.

Evidence supporting presence of two pacemakers in rat colon.

Intracellular microelectrodes and organ bath techniques were used to study spontaneous cyclic electrical and mechanical activity in the rat colon. Electron microscopy and immunohistochemical studies showed two major populations of interstitial cells of Cajal (ICC): one associated with Auerbach's plexus (ICC-AP) and one with the submuscular plexus (ICC-SMP). The ICC-SMP network partly adhered to the submucosa when removed and was generally strongly damaged after separation of musculature and submucosa. Similarly, longitudinal muscle removal severely damaged AP. Two electrical and mechanical activity patterns were recorded: pattern A, low-frequency (0.5--1.5 cycles/min), high-amplitude oscillations; and pattern B, high-frequency (13--15 cycles/min), low-amplitude oscillations. Pattern A was recorded in preparations with intact AP but absent in those without intact AP. Pattern B was recorded in preparations with intact SMP but was absent in those lacking SMP. With full-thickness strips, the superimposed patterns A and B were recorded in circular muscle. When longitudinal muscle mechanical activity was recorded, only pattern A was present. We conclude that two pacemakers regulate rat colonic cyclic activity: the ICC-SMP network (responsible for cyclic slow waves and small-amplitude contractions) and the ICC-AP network (which may drive the cyclic depolarizations responsible for high-amplitude contractions). This is the first report showing consistent slow wave activity in the rodent colon.

Op/op mice defective in production of functional colony-stimulating factor-1 lack macrophages in muscularis externa of the small intestine.

The osteopetrotic (op/op) mutant mouse possesses an inactivating mutation in the colony-stimulating factor-1 (CSF-1) gene, which results in the absence of certain macrophages and in osteopetrosis, following a lack of osteoclasts. Studies of the op/op mouse indicate that CSF-1-dependent tissue macrophages may belong to a trophic and/or scavenger subpopulation, which through their effect on other cell types can significantly affect tissue functions, and that cells which are CSF-1 independent have antigen presentation and immunological functions. We have previously identified a cell system of regularly distributed macrophages in the muscularis externa of the small intestine and wanted to extend these studies to the op/op mouse. The present investigations with light- and electron-microscopic methods using fluorescent dextran, methylene blue and immunohistochemistry (F4/80, anti-kit receptor, anti-CD3, anti-CD45R/B220) show that macrophages are absent from the muscle layers, with only an occasional macrophage present in the subserosa. In the lamina propria and submucosa, macrophage numbers are reduced. In all other respects the muscularis externa appears normal, including normal organization and number of interstitial cells of Cajal. Control and op/op mice both lack cells expressing CD3 (T lymphocytes), CD45R/B220 (B lymphocytes) and mast cells in the muscularis externa. This leaves the muscularis externa macrophages as the most likely source of local cytokine production under such conditions as postoperative ileus and intussusception in infants, where the muscularis externa appears to be one target of cytokines. We conclude that the lack of macrophages, combined with the preservation of otherwise normal structure, will make the op/op mouse a valuable model by which to assess the functions and relative importance of the muscularis externa macrophages in relation to intestinal motility under normal and pathological conditions.

c-kit immunoreactive interstitial cells of Cajal in the human small and large intestine.

c-kit immunohistochemistry was performed on unfixed frozen sections of human small (duodenum, jejunum, and ileum) and large intestine (ascending, transverse, descending, and sigmoid colon). The c-kit immunoreactive cells in the muscularis externa of the intestinal wall were identified as interstitial cells of Cajal (ICC) and mast cells. ICC were identified by their morphology, localization, and organization based on previous light and electron microscopic studies. In the small intestine, ICC were located primarily in relation to the myenteric plexus of Auerbach, but also in septa between circular muscle lamellae. In the large intestine, ICC were seen in relation to Auerbach's plexus, but also and in great numbers in the circular muscle layer and in teniae of the longitudinal muscle layer. The morphology of the ICC was similar in the small and large intestine, but the pattern of distribution was obviously different. c-kit immunoreactive mast cells were found predominantly in the inner part of the circular muscle layer. The anti-c-kit method is found to be an easy and reliable method to study at least most of the interstitial cells of Cajal and thereby contribute to further normal and pathological studies.

Action potential generation, Kit receptor immunohistochemistry and morphology of steel-Dickie (Sl/Sld) mutant mouse small intestine.

In contrast to wild-type mice, homozygotes with mutations of the W locus do not express the functional Kit receptor and are severely deficient in the Auerbach's plexus (AP)-associated subtype of interstitial cells of Cajal (ICC-AP). With a morphologically intact neural and muscular structure, the absence in these mutants of both small-intestinal slow waves and ICC-AP constitutes strong evidence for a key role of ICC-AP as pacemaker cells. In steel-Dickie mutant mice (Sl/Sld), the gene coding for the Kit ligand (stem cell factor) is defective. We examined Sl/Sld mutants and controls with intracellular microelectrode techniques, combined with light and electron microscopy. The absence of the normal Kit ligand (Sl/Sld mice) had very similar effects as the absence of the Kit receptor in viable mice, mutated at the White spotting, W, locus (W/Wv mice), in that neither slow waves, nor Kit receptor immunoreactivity in the region of Auerbach's plexus nor ICC-AP were present in the small intestine. In the Sl/Sld mouse, the smooth muscle cells generated action potentials at variable frequencies from a depolarized cell membrane of -40 to -55 mV. Increasing excitability by K channel blockers created many different patterns of action potential generation and the frequency increased from approximately 16 cpm to 66 cpm. This was in sharp contrast to control mice where action potentials were always restricted to the plateau phase of the slow waves and the slow wave frequency remained constant at approximately 39 cpm. Our data provide further strong support for the identification of ICC-AP as small-intestinal pacemaker cells. In addition, they provide a basis for the understanding of intestinal motor function without pacemaker activity.

Action potential generation in the small intestine of W mutant mice that lack interstitial cells of Cajal.

The small intestine of W/Wv mice lacks both the network of interstitial cells of Cajal (ICC), associated with Auerbach's plexus, and pacemaker activity, i.e., it does not generate slow-wave-type action potentials. The W/Wv muscle preparations showed a wide variety of electrical activities, ranging from total quiescence to generation of action potentials at regular or irregular frequency with or without periods of quiescence. The action potentials consisted of a slow component with superimposed spikes, preceded by a slowly developing depolarization and followed by a transient hyperpolarization. The action potentials were completely abolished by L-type Ca2+ channel blockers. W/Wv mice responded to K+ channel blockade (0.5 mM Ba2+ or 10 mM tetraethylammonium chloride) with effects on amplitude, frequency, rate of rise, and duration of the action potentials. In quiescent tissues from W/Wv mice, K+ channel blockade evoked the typical spikelike action potentials. Electron microscopy identified few methylene blue-positive cells in the W/Wv small intestine associated with Auerbach's plexus as individual ICC. Numbers of resident macrophage-like cells (MLC) and fibroblast-like cells (FLC) were significantly changed. Neither FLC nor MLC were part of a network nor did they form specialized junctions with neighboring cells as ICC do. Hence no cell type had replaced ICC at their normal morphological position associated with Auerbach's plexus. ICC were present in W/Wv mice at the deep muscular plexus in normal organization and numbers, indicating that they are not dependent on the Kit protein and do not take part in generation of pacemaker activity.

Macrophages in the external muscle layers of mammalian intestines.

The literature on macrophages in the muscularis externa of mouse, rat, guinea pig, cat, dog and human gut is reviewed. In smaller mammals macrophages are regularly situated in two locations: in the serosa and at the level of Auerbach's plexus between the longitudinal and circular muscle layers. In addition a few solitary cells are present at the level of the deep muscular plexus. At the level of Auerbach's plexus the macrophages occur as a constant and regularly distributed cell population with intimate associations between macrophages and interstitial cells of Cajal. Morphologically they differ from most resident macrophages in being irregular in shape with 4-6 primary cytoplasmic processes, which branch and give a stellate appearance. They have been demonstrated with endocytotic markers (trypan red, FITC-dextran, cholera toxin), immunocytochemically with macrophage antibodies (F4/80, M1/70) and antibodies against MHC class-II antigen, GABA and cGMP. In muscularis externa of the human gut a regularly distributed cell population of macrophages is not obvious. However, a phenotypically distinct subgroup is identified by light microscopy with the pan macrophage antibodies (EBM11, C3b1 and partly by p150.95), and shows MHC class-II antigen. By electron microscopy muscularis externa macrophages, in all species investigated, appear to be endocytically downregulated, and since they are lysozyme, prostaglandine H synthase (both constitutive and activated) and acid phosphatase negative, they appear to be inactivated cells. Both origin and function of these cells are unknown. They may be immuno-competent, participate in a neuroimmune axis, tissue growth and modulation or other regulations of specific cell functions.

W/kit gene required for interstitial cells of Cajal and for intestinal pacemaker activity.

The pacemaker activity in the mammalian gut is responsible for generating anally propagating phasic contractions. The cellular basis for this intrinsic activity is unknown. The smooth muscle cells of the external muscle layers and the innervated cellular network of interstitial cells of Cajal, which is closely associated with the external muscle layers of the mammalian gut, have both been proposed to stimulate pacemaker activity. The interstitial cells of Cajal were identified in the last century but their developmental origin and function have remained unclear. Here we show that the interstitial cells of Cajal express the Kit receptor tyrosine kinase. Furthermore, mice with mutations in the dominant white spotting (W) locus, which have cellular defects in haematopoiesis, melanogenesis and gametogenesis as a result of mutations in the Kit gene, also lack the network of interstitial cells of Cajal associated with Auerbach's nerve plexus and intestinal pacemaker activity.

Immunohistochemical localization of a gap junction protein (connexin43) in the muscularis externa of murine, canine, and human intestine.

Electron-microscopic studies have revealed a heterogeneous distribution of gap junctions in the muscularis externa of mammalian intestines. This heterogeneity is observed at four different levels: among species; between small and large intestines; between longitudinal and circular muscle layers; and between subdivisions of the circular muscle layer. We correlated results obtained with two immunomethods, using an antibody to the known gap-junctional protein (connexin43) with ultrastructural findings, and further evaluated the respective sensitivity of these two approaches. For comparative reasons we also included the vascular smooth muscle of coronary arteries into our study. Two versions of the immunotechnique (peroxidase-antiperoxidase and fluorescence methods) were applied to frozen sections of murine, canine, and human small and large intestines, as well as to pig coronary artery. In the small intestine of all three species a very strong reactivity marked the outer main division of the circular muscle layer, while the longitudinal muscle layer as well as the inner thin division of the circular muscle layer were negative. In murine and human colon both muscle layers were negative, while in canine colon the border layer between the circular muscle and the submucosa reacted strongly, and scattered activity was found in the portion of the circular muscle layer (one tenth of its thickness) closest to the submucosa. The remainder of the circular muscle layer and the entire longitudinal muscle layer were negative in the canine colon. In the coronary artery we could not confirm the positive, specific labeling reported by other investigators (l.c.).(ABSTRACT TRUNCATED AT 250 WORDS)

Ultrastructure of interstitial cells of Cajal in circular muscle of human small intestine.

BACKGROUND: Interstitial cells of Cajal (ICC) may be important regulatory cells in gut muscle layers. This study examined ICC within the circular muscle of human small intestine. METHODS: Surgically resected, uninvolved intestine was studied by light microscopy and electron microscopy. RESULTS: Muscle lamellae were separated by main septa in continuity with submucosa. Smooth muscle cells ran radially in the septa. Two types of ICC were distinguished. One ICC type had abundant intermediate filaments and smooth cisternae and a discontinuous basal lamina. This ICC type was present in the septa and in the outer third of the circular lamellae. The other ICC type had a complete basal lamina and conspicuous caveolae. This ICC type was observed only in the inner third of the circular lamellae. Both ICC types were close to nerves, but only the latter type formed gap junctions with one another and with muscle cells. Junctions between the two ICC types were not observed. CONCLUSIONS: The arrangement suggests that ICC and radially oriented muscle cells participate in electrical and mechanical coordination of the circular muscle layer of human small intestine.

Characterization of macrophage-like cells in the external layers of human small and large intestine.

In the external layers of human small and large intestine macrophage-like cells were characterized by immunohistochemical, histochemical and electron-microscopical methods. Using immunohistochemistry and a number of monoclonal antibodies, the presence and distribution of phenotypic subpopulations of macrophages were evaluated. In all locations macrophage-like cells were identified with antibody EBM11, which recognizes CD68 antigen, C3bi which recognizes CD11b, and partly with an antibody which recognizes protein 150,95 (CD11c). Macrophage-like cells in the external muscle layer were HLA-DR-positive (expressing the MHC class-II antigen), in contrast to macrophage-like cells in the subserosa and submucosa. Macrophage-like cells in the external muscle layer were mostly acid phosphatase-negative, and at the electron-microscopic level they were found to have features of macrophages: primary lysosomes, coated vesicles and pits. However, very few secondary lysosomes were present. Birbeck granules were not observed. It is concluded that in the external muscle layer of human small and large intestine numerous macrophages of a special type are present. It is discussed whether this cell type plays a role in gastrointestinal motility and/or has an immunological function.

Ultrastructure of interstitial cells of Cajal associated with deep muscular plexus of human small intestine.

Evidence showing that interstitial cells of Cajal have important regulatory functions in the gut musculature is accumulating. In the current study, the ultrastructure of the deep muscular plexus and associated interstial cells of Cajal in human small intestine were studied to provide a reference for identification and further physiological or pathological studies. The deep muscular plexus was sandwiched between a thin inner layer of smooth muscle (one to five cells thick) and the bulk of the circular muscle. Interstitial cells of Cajal in this region very much resembled smooth muscle cells (with a continuous basal lamina, caveolae, intermediate filaments, dense bodies, dense bands, and a well-developed subsurface smooth endoplasmic reticulum), but the arrangement of organelles was clearly different, and cisternae of granular endoplasmic reticulum were abundant. Interstitial cells of Cajal were distinguished from fibroblasts or macrophages in the region. They ramified in the inner zone of the outer division of circular muscle, penetrated the inner-most circular layer, and were also found at the submucosal border. They were in close, synapselike contact with nerve terminals of the deep muscular plexus, and only few gap junctions with other interstitial cells of Cajal or with the musculature were observed. Compared with interstitial cells of Cajal from other mammals, those associated with the deep muscular plexus in the human small intestine more closely resemble smooth muscle cells, and their organization appears more diffuse; however, the ultrastructure and organization of interstitial cells of Cajal is compatible with modulatory actions on the circular muscle also in humans.

Prostaglandin H synthase immunoreactivity in human gut. An immunohistochemical study.

Prostaglandins exhibit a variety of actions on intestinal smooth muscle depending upon the type, dose and muscle layer studied. As the cellular origin of prostaglandin H (PGH) synthase has not been established with certainty in the human gut wall, we studied the localization of PGH synthase in the human duodenum, jejunum, ileum and colon by immunohistochemistry. PGH synthase immunoreactivity appeared to be similar in all segments of the intestine. Most smooth muscle cells seemed to contain PGH synthase; however, the reaction in the lamina muscularis mucosae was much stronger than in the longitudinal and circular muscle layers. Endothelial cells in capillaries and larger vessels showed a positive reaction. In addition, unidentified cells in subserosa, at the level of Auerbach's plexus and in the submucosa were stained. We concluded that the smooth muscle cells of the human gut has a rather large capacity for PGH synthesis and the present results may provide a basis for a better understanding of both normal physiological functions as well as intestinal disease states involving disorders of prostaglandin synthesis.

Prostaglandin H synthase immunoreactivity localized by immunoperoxidase technique (PAP) in the small intestine and kidney of rabbit and guinea-pig.

Prostaglandins and inhibitors of prostaglandin synthesis have striking regulatory effects on intestinal muscularis externa. We suggested earlier that a population of macrophage-like cells, located between the external muscle layers might release prostaglandins with a local effect on enveloping interstitial cells of Cajal, postulated pacemaker cells of the gut. To determine cellular production site(s) of prostaglandin we applied monoclonal antibodies against prostaglandin H synthase combined with the PAP technique to sections of rabbit and guinea-pig small intestine and kidney. In rabbit small intestine muscle cells in the circular muscle layer and in the muscularis mucosae were positive, longitudinal muscle negative. Vascular endothelial cells and serosal mesothelial cells were stained. In guinea-pig all muscle layers were unstained but endothelial and mesothelial cells were stained together with unidentified cells in the outermost submucosa. In rabbit kidney, positive staining of collecting ducts, interstitial cells, the parietal layer of Bowman's capsule and arterial endothelial cells was present. Furthermore, we found prostaglandin synthase antigenicity in the epithelial cells lining the loop of Henle, not described before. In guinea-pig medullary collecting ducts were stained and the papilla was lined by stained epithelial cells. The results show a species variation in the distribution of recognizable levels of prostaglandin H synthase. The impressive reaction in the mesothelium must be considered, when enzyme distribution is examined biochemically with fractionated tissue. Our findings do not support our hypothesis that macrophage-like cells are more potent sources of prostaglandins than smooth muscle cells.