MCP-1 targeting inhibits muscularis macrophage recruitment and intestinal smooth muscle dysfunction in colonic inflammation

Upregulation of muscularis macrophage numbers and activities plays an important role in the intestinal dysmotility associated with intestinal inflammation. The present study aimed to clarify changes in population dynamics of intestinal muscularis macrophages during colonic inflammation and to test possible inhibitory actions of agents targeting monocyte chemoattractant protein-1 (MCP-1) on muscularis macrophage dynamics and motility disorder in the colonic inflammation elicited by 2,4,6-trinitrobenzene sulfonic acid. In the inflamed muscle layer, ED1 antibody-positive monocytes and monocyte-derived macrophages were increased, followed by increasing resident macrophages positively staining for ED2 antibody. Initiation of the ED1-positive macrophage dynamic is associated with MCP-1 mRNA expression. MCP-1 was expressed in both ED1- and ED2-positive macrophages after inflammation. Electromicroscopic analysis revealed that the cell-division phase of muscularis macrophages was seen only in the early stages of inflammation. In addition, ED1 and ED2 double-positive macrophages can be detected during inflammation. Treatment with dominant negative MCP-1 or neutralizing MCP-1 antibodies markedly inhibited numbers of both ED1- and ED2-positive macrophages. Inflammation-mediated dysmotility was partially recovered by treatment with neutralizing MCP-1 antibodies. These results suggest that the inflamed muscle layer is initially infiltrated by monocytes, which then differentiate and develop into muscularis-resident macrophages. These macrophages express MCP-1 for further recruitment of monocytes. MCP-1 may be one potential therapeutic target for inhibiting intestinal motility disorders in gut inflammation.     

The gamma 2 subunit is an integral component of the gamma-aminobutyric acidA receptor but the alpha 1 polypeptide is the principal site of the agonist benzodiazepine photoaffinity labeling reaction

Polyclonal antibodies were raised to a synthetic peptide whose amino acid sequence was derived from the novel gamma-aminobutyric acidA (GABAA) receptor subunit, gamma 2. These anti-gamma 2 1-15 Cys antibodies reacted specifically with the GABAA receptor purified from adult bovine cerebral cortex in an enzyme-linked immunosorbent assay. Anti-gamma 2 1-15 Cys antibodies specifically immunoprecipitated [3H]flunitrazepam photoaffinity-labeled native receptor in parallel with anti-alpha 1 324-341 antibodies. Immunoprecipitation of sodium dodecyl sulphate (SDS) denatured photoaffinity-labeled receptor by anti-gamma 2 1-15 Cys antibodies, however, resulted in a significant decrease in the maximum percentage of radioactivity immunoprecipitated compared to that by anti-alpha 1 324-341 antibodies. In immunoblots, anti-gamma 2 1-15 Cys antibodies reacted with a broad band in the molecular weight range Mr 43,000-49,000 which was distinct from that recognized by anti-alpha 1 324-341 antibodies. The anti-alpha 1 324-341 immunoreactive band was the main subunit irreversibly photoaffinity labeled by [3H]flunitrazepam, i.e. Mr 53,000. These results demonstrate for the first time that the gamma 2 subunit is an integral component of the GABAA receptor but it is the alpha 1 subunit that is the principal site of the agonist benzodiazepine photoaffinity labeling reaction. It supports a role of both the alpha 1 and gamma 2 polypeptides in the formation of the central benzodiazepine binding site within a GABAA receptor oligomer.     

An immunocytochemical study of keratin reactivity during rat odontogenesis

Summary The cytokeratin distribution in the developing rat enamel organ from day 15 of gestation through to 11 days post partum was examined immunohistochemically using a panel of monoclonal antibodies. A temporo-spatial programme of keratin expression was observed during odontogenesis and positive reactivity of the enamel organ was seen with the pan keratin antibodies CK1 (clone LP34 — reacts with a number of keratins including 6 and 18) and AE1-3 (reacts with most acidic and basic keratins). No reactivity was observed in the enamel organ with the other antibodies examined (Ks 8.12 [reacts with keratins 13 and 16], Ks 8.60 [reacts with keratins 10 and 11) and MCA157 [reacts with rat liver antigen]), although these antibodies did stain other epithelial tissues. This study supports the view that the epithelial cells of the enamel organ synthesize a tissuspecific subset of keratins which are related to the differentiation of the cells.     

Class II MHC-expressing cells in the rat adrenal gland defined by monoclonal antibodies

 The detailed distribution and heterogeneity of various immunocompetent cells were characterized in the normal adrenal gland of the rat, with special emphasis on major histocompatibility complex (MHC) class II-expressing cells and macrophages. All adrenals contained at least two different populations of cells reactive with the dendritic cell or the macrophage antibodies. These cells were clearly distinguished from adrenal parenchymal cells by their morphology and location. The majority of dendritic cells were immunoreactive for the MHC class II (Ia) antigen (MRC OX6) and/or the dendritic cell antibodies (MRC OX62), and negative for the macrophage antibodies (ED1, ED2, and/or MRC OX42), whereas the main population of macrophages was immunonegative for the former antibodies and positive for the latter. The OX62-positive cells and the OX42-labeled cells occurred exclusively throughout the medulla. The cellular density of dendritic cells in the adrenal cortex was significantly higher than that of macrophages. Double-immunoperoxidase staining for ED1 and OX6 revealed that positively stained cells could be classified into the following categories: ED1⁺OX6⁺, ED1⁺OX6⁻, and ED1⁻OX6⁺. More then 40% of OX6⁺ cells were immunoreactive for ED1 in the zona glomerulosa, while approximately 15%, 20%, and 30% of OX6⁺ cells were positive for ED1 in the zona fasciculata, zona reticularis and medulla, respectively. ED1⁺ED2⁻ cells were more frequently detected in the zona glomerulosa than in other adrenal zones. Only a few ED1⁻ED2⁺ cells were located in the zona glomerulosa, whereas a large number of them were found in the zona fasciculata. In the zona reticularis and medulla, ED1⁺ED2⁺, ED1⁺ED2⁻, and ED1⁻ED2⁺ cells were detected in the ratio 2:1:3. Our rsults suggest that dendritic cells and macrophages mature during their migration within the adrenal gland. These immunocompetent cells may contribute to a paracrine regulation of adrenal function under physiological conditions. Accepted: 3 November 1997     

Monoclonal antibodies specific to porin of Haemophilus influenzae type b: localization of their cognate epitopes and tests of their biological activities

The major outer membrane protein of Haemophilus influenzae type b (Hib) is porin (Mr 38,000, 341 amino acids). To identify antigenic determinants on Hib porin that might be exposed at the bacterial cell surface, seven mouse monoclonal anti-Hib porin antibodies were generated. The monoclonal antibodies were tested for their binding to intact cells by flow cytometry; all but one bound to the cell surface. Digestions of Hib porin with cyanogen bromide, hydroxylamine or trypsin generated fragments, the identities of which were confirmed by microsequencing of the amino termini. Following electrophoresis and immunoblotting of the fragments, the specificities of the monoclonal antibodies for their cognate sequences were determined. The porin gene ompP2 was expressed in the baculovirus expression vector system; the recombinant porin was recognized by all of the monoclonal antibodies. Deletions were created by omega mutagenesis of ompP2, generating proteins truncated after amino acids 139, 174, 182, and 264. These deletion proteins were tested for reactivities with the monoclonal antibodies, thereby establishing the boundaries of three antigenic determinants that were recognized by the monoclonals: domain (i), amino acids 104-139; domain (ii) amino acids 162-174; and domain (iii), amino acids 267-341. The biological activities of monoclonal antibodies that were representative of these three classes were tested for their bactericidal activity in complement-mediated lysis of whole cells. The monoclonal antibodies were also tested for their immunoprotective properties in the infant rat model of bacteraemia. Although the monoclonal antibodies were surface-binding, they were neither bactericidal nor protective.     

Detection of a soluble serum antigen by a new monoclonal antibody ED8 in patients with breast cancer

A murine hybridoma, secreting monoclonal antibody ED8, was generated by immunization with the breast cancer cell line H466B. ED8 reacts with a 300 kDa antigen present on H466B cell surface. The antigen can be detected in serum from breast cancer patients, using ELISA and immunoblotting.     

Delineation of epitopes on the Py235 rhoptry antigen of Plasmodium yoelii YM

The 235-kDa antigenic rhoptry protein Py235 of Plasmodium yoelii is encoded by a large, highly polymorphic gene family. Monoclonal antibodies to some of these antigens have been shown to attenuate the virulence of the lethal YM strain of the parasite, converting a potentially fatal YM infection to a fulminating one typical of the nonlethal 17X strain, by inducing a switch in target cell preference from mature red blood cells to reticulocytes. The reason for this is not known but would suggest that antigenic determinants of Py235 may be useful in or as subunit vaccines. To identify such determinants, we constructed an epitope expression library of one Py235 variant and screened the library with the antibodies. Thus, we mapped 5- and 12-amino acid epitopes to the C-terminus of the antigen. Both epitopes were more reactive with protective than with nonprotective monoclonal antibodies. This may explain the differential protection conferred by these antibodies upon their passive transfer into mice.     

Phagocytic Cells in the Taste Buds of Rat Circumvallate Papillae after Denervation

Phagocytic cells in the taste buds of rat circumvallate papillaeafter the sectioning of bilateral glossopharyngeal nerves wereexamined by electron microscopy and immunohistochemistry. Electronmicrographs taken 1 day after denervation revealed that flat-shapedcells were present just beneath the taste buds and that theircellular processes extended toward the debris from the degeneratingtaste buds. At 2–6 days after denervation, long and thinprocesses of the flat cells surrounded the debris and appearedto have taken them up into the cytoplasm as small vesicles.Evidence for phagocytosis by the flat cells was seen up to 9days after denervation and again at 24 and 40 days, in correlationto the degeneration and regeneration of the taste buds. Pre-embeddingimmunohistochemistry using anti-vimentin antibody showed thatflat cells strongly reacted with vimentin. Light microscopicimmunohistochemistry using anti-macrophage antibodies (ED1,ED2) showed that throughout the post-operative days macrophageswere not present underneath or within the taste buds. Most ofthe ED2-immunoreactive resident macrophages were located inthe deep layer of connective tissue, and a few were found inthe nerve bundle. ED1-immunoreactive cells were seen in theduct cells of von Ebner's glands and a few were in the trenchwall of circumvallate papillae; however, they were also immunoreactivefor anti-OX62 antibody, which recognizes dendritic cells. Theresults indicate that the phagocytic cells of the taste budsare fibroblasts, not macrophages. Moreover, resident macrophagesparticipate in phagocytosis of degenerated nerves together withSchwann cells. Chem. Senses. 21: 467–476, 1996.     

Morphogenesis of rat cranial meninges

Summary The meninges of albino Wistar rat embryos, aged between the 11th embryonic day (ED) and birth, were sectioned using a specially constructed device. This technique permits optimal microanatomical preservation of all tissues covering the convexity of the brain: skin, muscle, cartilage or bone, and the meninges. At ED11, the zone situated between the epidermis and the brain is occupied by a mesenchymal network. At ED12, part of this delicate network develops as a dense outer cellular layer, while the remainder retains its reticular appearance, thus forming an inner layer (the future meningeal tissue). At ED13, the dura mater starts to differentiate. At ED14, the bony anlage of the skull can be identified, and along with the proceeding maturation of dura mater some fibrillar structures resembling skeletal muscle fibers appear in the developing arachnoid space. At ED15–17, a primitive interface zone — dura mater/ arachnoid — is formed, comprised by an outer electronlucent and an inner electron-dense layer marking the outer aspect of the arachnoidal space. At ED18–19, the innermost cellular row of the inner durai layer transforms into neurothelium, which is separated from the darker arachnoidal cells by an electron-dense band. The arachnoidal trabecular zone with the leptomeningeal cells is formed at ED19. By the end of the prenatal period (ED20–21), its innermost part organizes into an inner arachnoidal layer and an outer and inner pial layer. The results from this study indicate (i) that dura mater and leptomeninges develop from an embryonic network of connective tissue-forming cells, and (ii) that the formation of cerebrospinal fluid (CSF)-containing spaces accompanies the differentiation of the meningeal cellular layers.     

Reticulum cells in the ontogeny of nasal-associated lymphoid tissue (NALT) in the rat

This study concerns the ontogeny of reticulum cells (RC) in the nasal-associated lymphoid tissue (NALT) of Wistar and Brown-Norway rats. A panel of monoclonal antibodies (mAb) directed against RC in peripheral lymphoid organs (antibodies ED10ED15) was used, together with a recently developed antibody ED17, which recognizes macrophages and Langerhans cells. Early in embryogenesis, staining with common connective tissue markers, ED14 and ED15, was found. ED17-positive cells were present before cells positive to ED1, a pan-macrophage marker, or Ia glycoproteins were observed. The first differentiation of reticulum was seen at the day of birth, when ED10 recognized a distinct area in the nasal mucosa. The first T-lymphocytes were found at the same time. Two days after birth, B-cells and ED11-positive cells were present in the NALT area. Fourteen days after birth, T- and B-cell compartments were recognizable. ED10 was found predominantly in the T-cell area and ED11 was mainly confined to the B-cell compartment. We conclude that the development of the NALT is closely accompanied by the phenotypic specialization of the reticulum. This suggests that the reticulum plays an important role in the compartmentalization of NALT tissue and in the retention of lymphocyte subsets within these compartments.     

Three Distinct Subsets of Thymic Epithelial Cells in Rats and Mice Defined by Novel Antibodies

Aim

Thymic epithelial cells (TECs) are thought to play an essential role in T cell development and have been detected mainly in mice using lectin binding and antibodies to keratins. Our aim in the present study was to create a precise map of rat TECs using antibodies to putative markers and novel monoclonal antibodies (i.e., ED 18/19/21 and anti-CD205 antibodies) and compare it with a map from mouse counterparts and that of rat thymic dendritic cells.

Results

Rat TECs were subdivided on the basis of phenotype into three subsets; ED18⁺ED19^+/−keratin 5 (K5)⁺K8⁺CD205⁺ class II MHC (MHCII)⁺ cortical TECs (cTECs), ED18⁺ED21⁻K5⁻K8⁺ Ulex europaeus lectin 1 (UEA-1)⁺CD205⁻ medullary TECs (mTEC1s), and ED18⁺ED21⁺K5⁺K8^dullUEA-1⁻CD205⁻ medullary TECs (mTEC2s). Thymic nurse cells were defined in cytosmears as an ED18⁺ED19^+/−K5⁺K8⁺ subset of cTECs. mTEC1s preferentially expressed MHCII, claudin-3, claudin-4, and autoimmune regulator (AIRE). Use of ED18 and ED21 antibodies revealed three subsets of TECs in mice as well. We also detected two distinct TEC-free areas in the subcapsular cortex and in the medulla. Rat dendritic cells in the cortex were MHCII⁺CD103⁺ but negative for TEC markers, including CD205. Those in the medulla were MHCII⁺CD103⁺ and CD205⁺ cells were found only in the TEC-free area.

Conclusion

Both rats and mice have three TEC subsets with similar phenotypes that can be identified using known markers and new monoclonal antibodies. These findings will facilitate further analysis of TEC subsets and DCs and help to define their roles in thymic selection and in pathological states such as autoimmune disorders.     

Resident macrophages (ED2- and ED3-positive) do not phagocytose degenerating rat skeletal muscle fibres

Two soleus muscles with degenerating muscle fibres were serially sectioned and adjacent sections from various levels of the skeletal muscles were stained with antibodies that react with either monocytes and inflammatory macrophages (ED1) or with the major subpopulations of resident macrophages (ED2 and ED3). ED2⁺ and ED3⁺ resident macrophages were abundant throughout the muscles but were not present within the degenerating fibres. ED1⁺ cells, in contrast, were rarely observed within the undamaged regions of the muscles but were abundant within the degenerating fibres and in the perimysium between arterioles and degenerating fibres. It is concluded that the phagocytosis of damaged muscle fibres is not carried out by the major subpopulations of resident macrophages.     

Characteristics of the reaction of healthy human blood serum with the epidermis

Indirect immunofluorescence has shown that the blood serum of normal subjects reacts with cytoplasm antigens of epidermis differentiated cells in 100% of the cases. The level of antibodies and the immunomorphological picture of the reaction are marked by high constancy and intense fluorescence of the cytoplasm of epidermis differentiated cells, while the reaction with basal layer cell antigens is observed comparatively seldom and little pronounced. The authors discuss possible participation of antibodies contained by the normal blood serum and of their complexes with tissue antigens in the regulation of vital activity of the cells and immune response to the host antigens.     

Biochemical evidence for the existence of gamma-aminobutyrateA receptor iso-oligomers

Polyclonal antibodies were raised against synthetic peptides whose sequences were from unique regions of the bovine gamma-aminobutyrateA receptor alpha 1, alpha 2, and alpha 3 subunits. The anti-alpha 1 324-341, anti-Cys alpha 2 414-424, and anti-Cys alpha 3 454-467 antibodies all specifically immunoprecipitated [3H]flunitrazepam and [3H]muscimol binding activities in parallel from Na+ deoxycholate extracts of bovine cerebral cortex. The maximum number of benzodiazepine binding sites immunoprecipitated by each antibody in three brain regions, cerebral cortex, cerebellum, and hippocampus, was investigated. Differences were found for both the maximum number of sites immunoprecipitated by each antibody in one brain region and for the percentage of benzodiazepine binding sites immunoprecipitated by one specificity antibody between the different brain regions. Furthermore, it was found that co-immunoprecipitation with either anti-alpha 1 324-341, anti-Cys alpha 2 414-424, and anti-Cys alpha 3 454-467 or anti-alpha 1 324-341 and anti-Cys alpha 3 454-467 antibodies resulted in an increase in the percentage of benzodiazepine binding sites immunoprecipitated, the sum of which was equal to the percentages pelleted by the individual antibodies. These results demonstrate for the first time the existence in mammalian brain of gamma-aminobutyrateA receptor alpha subunit iso-oligomers.     

Role of Ser-340 and Thr-341 in transmembrane domain IX of the Na+/proline transporter PutP of Escherichia coli in ligand binding and transport

The Na+/solute symporter family comprises more than 400 members of pro- and eukaryotic origin. Using the Na+/proline transporter PutP of Escherichia coli as a model, the role of two conserved residues, Ser-340 and Thr-341, is investigated to obtain insights into the mechanism of transport catalyzed by members of this family. Substitution of these amino acids alters the transport kinetics of cells and proteoliposomes containing the PutP variants significantly. In particular, the apparent affinities for Na+ and Li+ are reduced by 2 orders of magnitude or more. Also proline binding is affected, albeit to a lesser extent than ion binding. Thereby, the presence of a hydroxyl group at position 341 is essential for high affinity ligand binding. Furthermore, Cys placed at position 340 or 341 reacts with sulfhydryl reagents of different polarity, indicating accessibility from the water phase. In addition, Cys cross-linking suggests proximity of the residues to other amino acids previously shown to be crucial for ligand binding. For these reasons it is suggested that Ser-340 and Thr-341 are located in a ligand translocation pathway. Furthermore, it is proposed that the side chain of Thr-341 directly participates in Na+ binding.     

An immunocytochemical study of keratin reactivity during rat odontogenesis.

The cytokeratin distribution in the developing rat enamel organ from day 15 of gestation through to 11 days post partum was examined immunohistochemically using a panel of monoclonal antibodies. A temporo-spatial programme of keratin expression was observed during odontogenesis and positive reactivity of the enamel organ was seen with the pan keratin antibodies CK1 (clone LP34 - reacts with a number of keratins including 6 and 18) and AE1-3 (reacts with most acidic and basic keratins). No reactivity was observed in the enamel organ with the other antibodies examined (Ks 8.12 [reacts with keratins 13 and 16], Ks 8.60 [reacts with keratins 10 and 11) and MCA157 [reacts with rat liver antigen]), although these antibodies did stain other epithelial tissues. This study supports the view that the epithelial cells of the enamel organ synthesize a tissue-specific subset of keratins which are related to the differentiation of the cells.     

Laminar restriction of retinal macrophagic response to optic nerve axotomy in the rat

Following optic nerve transection, most of the retinal ganglion cells die. Their debris is promptly cleared by phagocytic cells. It is currently not known to what extent peripherally derived macrophages contribute to this activity. Using antibodies OX42 and ED‐1, phagocytic cells were labeled in the retinas of optic nerve lesioned adult rats. To distinguish whether the cells were reactive microglial or macrophagic in origin, blood‐borne monocytes were labeled with fluorescent microspheres while in the systemic circulation. Macrophages invaded the retina, but only in the nerve fiber layer, sparing the ganglion cell and other layers. These macrophages engulfed only the axonal debris from dying ganglion cells, not their degenerating cell bodies. These results indicate that although peripherally derived monocytic cells are recruited into the retrogradely degenerating retina, their role in clearing debris is limited to the optic fiber layer. © 1999 John Wiley & Sons, Inc. J Neurobiol 40: 55–66, 1999     

Ontogeny of macrophage subpopulations and Ia-positive dendritic cells in pulmonary tissue of the rat

Summary The development of macrophage subpopulations and dendritic cells in the rat lung was studied from day 15 of gestation until day 21 after birth by means of immunohistochemical techniques combined with acid phosphatase staining. To characterize these cell populations, monoclonal antibodies raised against rat macrophage subpopulations were used (ED1, ED2, ED7, and ED8) in addition to anti-Ia antibodies. Ia-positive cells with a dendritic morphology were found on day 16 of gestation. During ontogeny, the number of these cells gradually increased. They were always found in mesenchymal lung tissue between the epithelial tubules of future alveoli, and in perivascular or peribronchial areas. ED1-positive macrophages were found on day 17 of gestation, with a distribution different from that of Ia-positive dendritic cells. The distribution of ED1-positive cells changed during ontogeny: before birth, ED1-positive cells were present in mesenchymal areas of lung tissue, whereas after the first week of postnatal life ED1 recognized all free alveolar macrophages. No Ia-expression was found on free alveolar macrophages. This developmental pattern resembles the ontogeny of Ia-positive dendritic cells and ED1-positive macrophages in gutassociated tissue. The comparable development of these cell populations in gut and lung tissue indicates a common ontogeny in the mucosal immune system.Fellow of the Royal Netherlands Academy of Arts and Sciences     

Protective antigens of rodent and human bloodstage malaria

Bloodstage malaria parasites are antigenically complex, but individual antigens can be identified and analysed using monoclonal antibodies. Two monoclonal antibodies that recognize a 235 000 molecular mass Plasmodium yoelii rhoptry protein provide some protection when injected into mice against a challenge infection. The purified rhoptry protein also provides protective immunity against P. yoelii YM when used to vaccinate mice and fulminating infections are converted into self-limiting, reticulocyte-restricted infections. Another monoclonal antibody immunoprecipitates a 230 000 molecular mass protein and a series of proteolytic processing fragments. At least one of these processing fragments, probably a 90 000 molecular mass species, is located on the merozoite surface. Mice immunized with the purified protein were protected against challenge infection with P. yoelii YM. This antigen may provide protection by inducing a cell-mediated immune response. A monoclonal antibody raised against P. falciparum schizonts reacts with a 195 000 molecular mass protein which is synthesized in schizonts and subsequently cleaved. Fragments of the 195 000 molecular mass protein are expressed as major antigens on the merozoite surface. The 195 000 molecular mass P. falciparum protein and the 230 000 molecular mass P. yoelii protein belong to a class of malaria parasite antigens which probably is important in the induction of a protective immune response in the host.     

Glycosyl receptors in macrophage subpopulations of rat spleen and lymph node

Summary We have developed an immunohistochemical method for the in vivo and in vitro detection of glycosyl receptors in rat spleen and lymph nodes by using neoglycoproteins. The receptor in both organs recognized mannose coupled to bovine serum albumin (mannose-BSA), fuscose-BSA, N-acetylglucosamine-BSA and to a lesser extent glucose-BSA, but not galactose-BSA or N-acetylgalactosamine-BSA. In vitro neoglycoprotein-receptor binding was Ca²⁺ dependent and could be inhibited by mannan but not by mannose. Simultaneous staining with the monoclonal antibodies ED1, ED2 or ED3 revealed that only ED1-and ED3-positive macrophages were involved in the binding of neoglycoproteins. In the spleen, the marginal-zone macrophages and a subpopulation of the marginal metallophils possess glycosylbinding receptors. In the lymph nodes, the medullary sinus macrophages and a subpopulation of the outercortex macrophages are able to bind neoglycoproteins.