Lipocortin-1 Immunoreactivity Research Articles

Microglia in organotypic hippocampal slice culture and effects of hypoxia: ultrastructure and lipocortin-1 immunoreactivity

Lipocortin-1 immunocytochemistry was used to study the various cell forms of microglia that appear during organotypic hippocampal tissue culture, as well as in the in vitro toxic hypoxia model. Antibodies against lipocortin-1 identified activated and phagocytic cells that were abundant in a slice after the plating of a culture: cells of the intermediate form at the later time-points of culturing, resting ramified microglia beginning from the seventh day of culturing, as well as activated and phagocytic cells that appeared in the slice after experimental toxic hypoxia induced by potassium cyanide treatment. Lipocortin-1-positive microglia cell forms corresponded well to the description of the microglia in vivo, and the morphology of microglia corresponded to the circumstances under which these cells were observed in slice cultures. Electron microscopic studies have demonstrated, for the first time, that microglia in organotypic slice culture preserve morphological features typical of different microglial forms in vivo, as well as specific contacts and interactions with the other neural tissue elements. After experimental toxic hypoxia, rapid changes in microglial ultrastructure and localization were observed, reminiscent of in vivo models of ischaemia. In conclusion, observations of microglial morphology and behaviour allow us to suggest that microglia in the organotypic culture preserve their essential characteristic features and properties, thus providing an important model system for studying the structure and function of these cells.

Increased lipocortin-1 (annexin-1) expression in the sciatic nerve of Lewis rats with experimental autoimmune neuritis.

Lipocortin-1 exerts a potent immunosuppressive effect on pathogenic T cells. In multiple sclerosis and experimental autoimmune encephalomyelitis levels of lipocortins are raised, suggesting their involvement in the recovery from an immunological insult or in neural regeneration. To further understand the role of lipocortins in the peripheral nervous system we have characterized lipocortin-1 levels and cellular distribution of lipocortin-1 immunoreactivity in sciatic nerves of rats with experimental autoimmune neuritis (EAN), a model of human Guillain-Barré syndrome. EAN was induced actively by immunization with bovine peripheral myelin (active EAN) or by adoptive-transfer (AT-EAN) of P2-specific T cells. Cellular infiltrates in serial and semithin cryosections were characterized by immunohistochemistry. In parallel, lipocortin-1 levels in tissue extracts were quantified by a sandwich-ELISA. Only weak lipocortin-1 immunoreactivity was found in nerves of control animals injected with non-pathogenic T cells. The majority of macrophages and lymphocytes in EAN lesions exhibited lipocortin-1 immunoreactivity. Some very heavily stained cells showed a distribution and morphology similar to ED-2-positive macrophages which were abundant during early stages of EAN. Lipocortin-1 expression in T cells and macrophages was proven by immunocytochemical studies in semithin serial sections. In tissue extracts, lipocortin-1 levels increased from 0.24 +/- 0.14 micrograms/mg protein in controls receiving non-pathogenic T cells to a maximum of 0.55 +/- 0.1 micrograms/mg protein in AT-EAN at the peak of disease, and then slowly decreased during clinical recovery but still remained elevated. In dose-response studies in AT-EAN, highest values of lipocortin-1 (0. 71 +/- 0.23 micrograms/mg protein) were recorded after transfer of 2 x 10(7) T cells. Increased levels of lipocortin-1 were also measured in active EAN but occurred during the recovery phase (0.65 +/- 0.27 micrograms/mg protein). By analogy with other immune-mediated disorders, increased lipocortin-1 expression in the inflamed sciatic nerve in EAN may exert immunoregulatory functions in-situ and contribute to the termination of the autoimmune response.

Lipocortin 1 (annexin 1): a candidate paracrine agent localized in pituitary folliculo-stellate cells.

It is now well established that lipocortin 1 (LC1) plays an important role as a mediator of early delayed glucocorticoid feedback action in the hypothalamo-hypophysial system. In both the hypothalamus and anterior pituitary gland, LC1 mimics some of the actions of glucocorticoids; moreover, glucocorticoids stimulate the synthesis of LC1 and cause the translocation of intracellular LC1 to the outer cell surface. The mechanism by which LC1 acts in these tissues is only partially understood, but may involve paracrine and/or autocrine actions. To address these possibilities we have investigated the localization of LC1 in the rat pituitary gland, using double labeling immunohistochemistry to identify the pituitary cell types that express LC1. At the light microscopic level LC1 was not detected in the endocrine cells in cryosections of the pituitary, but it was found in abundance in the surrounding folliculo-stellate (FS) cells. In the anterior and interme diate pituitary lobes, there was a near total colocalization of LC1 and S100, a specific marker of FS cells. By contrast, in the posterior pituitary gland, LC1 immunoreactivity was not colocalized with S100 which labeled most pituicytes, or with OX-42 monoclonal antibody, a marker of the microglial cells. Immunogold electron microscopy confirmed that LC1 is present in the nongranulated FS cells. LC1 im munoreactivity was also present in a mouse pituitary FS-like cell line (TtT/GF), particularly in the periphery of the cytoplasm. The localization of LC1 in the FS cells of the anterior pituitary gland defines LC1 as a new marker of the FS cell population. These results support our hypothesis that LC1 acts as one of the paracrine agents liberated by FS cells that modulate the release of pituitary hormones.

De novo expression of lipocortin-1 in reactive microglia and astrocytes in kainic acid lesioned rat cerebellum.

An understanding of the role of reactive glia in the neurodegenerative/regenerative process requires a knowledge of the molecules synthesised by these cells following trauma. We investigated the cellular localisation of lipocortin-1 (LC-1), a putative neuroprotective agent, in cryostat sections of normal and kainic acid lesioned rat cerebellum. In the normal cerebellum lipocortin-1 immunoreactivity was detected in Purkinje cell bodies and molecular layer interneurons. Following kainic acid (1 microg) induced lesions, it was rapidly upregulated in activated microglia, from which it appeared to be secreted. At later time points it was detected in activated astrocytes. LC-1 protein levels were quantified by a sensitive and specific ELISA. Compared to control cerebellum, LC-1 levels were dramatically elevated following lesion, peaking at 3 days: 760% of basal (unlesioned) levels. In situ hybridisation studies revealed a marked upregulation of LC-1 mRNA at 1 and 3 days following the lesion, indicating the transient de novo synthesis of this protein, consistent with a localisation to microglia. In vitro studies, on cultured astrocytes and microglia, demonstrated high levels of intracellular LC-1 in both cell types. LC-1 was detected in microglial but not astrocytic, conditioned media, confirming the in vivo observations that activated microglia may secrete LC-1. Our data show that at early time points following excitotoxic lesion to the cerebellum, it is activated microglia that synthesise and possibly secrete this protein, suggesting an important role of this cell type in immunosuppression and neuroprotection following damage to the central nervous system.

Gliogenesis in postnatal rat optic nerve: LC1+ microglia and S100- β+ astrocytes

Lipocortin 1 (LC1) and S100- β, two Ca 2+-binding proteins that serve as specific markers for microglia and astrocytes, respectively, have been used to study postnatal gliogenesis in the rat optic nerve. Computerized image analysis was used to quantify and map the stained and unstained glia in transverse sections (10 μm thick) taken 1–2 mm from the chiasm in optic nerves from rat pups at postnatal day 0 (P0), P7, P14, P21, P28, P38 and adults. The number of astrocytes was remarkably constant (100 per section) at all ages. Because the area of the nerve increases 10-fold from P0 to adult, the population density of astrocytes begins at >5000 mm −2 and drops to 400 mm −2 in the mature nerve; however, because the nerve length increases two-fold, the number of astrocytes doubles over the same period. In contrast, the number of LC1+ cells per section initially is sparse (4 at P0), increases rapidly up to 36 at P21 and levels off at 49 in adults. The microglia population density is relatively stable throughout development (200–300 mm −2) except during the peak of oligodendroblast apoptosis (P21) when it rises to 450 mm −2. Neonatally, LC1 immunoreactivity predominantly labels spherical-ameboid cells; but by P28 they are replaced by mature ramified microglia. The number of unstained cells (putative oligodendrocytes) per section increases from 11 at P0 to a peak of 308 at P21, and declines slightly to 269 in adults. While generally confirming concepts of astrocyte and oligodendrocyte ontogeny from the literature, the present report adds considerable detail regarding microglia, which often have been ignored. Microglia identified by LC1 immunoreactivity comprise 12% of the glia in adult optic nerve near the chiasm.

Infraorbital nerve transection and whisker follicle removal in adult rats affect microglia and astrocytes in the trigeminal brainstem. A study with lipocortin1- and S100 β-immunohistochemistry

Transections of the infraorbital nerve in adult rats resulted in progressive alterations of microglia identified by Lipocortin1 immunoreactivity at the sites where the primary afferents terminate, i.e. in the trigeminal brainstem sensory nuclei. Microglia proliferated three- to four-fold. Their cell bodies enlarged and their processes thickened. Microglial responses were similar to the removal of whisker follicles. However, they were restricted to discrete nuclear subregions that matched with the known whisker somatotopy. Astrocytes identified by S100 β immunoreactivity showed minor increases in size and in population density. No microglial or astrocytic reactions were found in the second and third synaptic relays of the somatosensory pathway. Because both types of lesion reportedly lead to the reorganization of primary afferents, our results establish the two experimental designs as valuable tools to elucidate the role of microglia and Lipocortin1 in adult brain plasticity.

Lipocortin 1 in apoptosis: mammary regression.

Enigmatically, degradation of debris generated in programmed cell death (apoptosis) elicits little inflammation. Having previously detected the upregulation of lipocortin 1 (LC1), a 35-kDa protein with anti-inflammatory and immuno-suppressive properties, at sites of non-inflammatory phagocytosis in the central nervous system (J Neurosci Res 36:491-500, 1993), we sought to determine if LC1 was involved in apoptosis. LC1 immunoreactivity in mammary glands of adult rats was quantified in situ using video microdensitometry before and during postlactational regression. LC1 is present in the mammary ducts but is absent from the alveoli during lactation. One day after weaning, however, LC1 is detected in the lactiferous cells and, as apoptosis proceeds over the ensuing 4 days, total LC1 in the gland increases > 10-fold over resting levels. LC1 remains high in both the apoptotic cells and epithelial phagocytes through day 10, but the total LC1 per gland drops as the apoptotic cells are cleared. Published experiments have shown that LC1 specifically binds Ca++ and phosphatidylserine, and that these affinities are modulated by tyrosine phosphorylation and cross-linking with transglutaminase. Thus, LC1 appears to be a candidate for several putative activities in apoptosis (e.g., phagocyte recognition via phosphatidylserine binding and/or buffering intracellular Ca++) in addition to its anti-inflammatory role.

Cytokines, prostaglandins and lipocortin-1 are present in the brains of scrapie-infected mice

The presence of cytokines, prostaglandins and lipocortin-1 was investigated in terminally affected mice in two models of scrapie. There was marked induction of glial interleukin-1β, tumour necrosis factor α, prostaglandin E 2, prostaglandin F 2α and lipocortin-1 immunoreactivity in those areas of the brain showing the characteristic vacuolation of scrapie. A comparison of these staining patterns with those of GFAP and F4/80 showed that their expression occurred predominantly in astrocytes. It is possible that cytokines play a significant role in the pathogenesis of neurodegeneration in scrapie.

Lipocortin-1 immunoreactivity in the human pituitary gland.

We evaluated the distribution of lipocortin-1 immunoreactivity in 118 immature or mature human hypophyses using the peroxidase-antiperoxidase (PAP) technique with a polyclonal rabbit antiserum against lipocortin-1. Serial sections were evaluated for five pituitary hormones and S-100 protein immunoreactivity to compare their distributions with that of lipocortin-1. Scattered or moderate numbers of cells exhibited lipocortin-1 immunoreactivity in the pars distalis of 89 subjects ranging in age from 27 weeks' gestation to 83 years. Seven immature and seven aged specimens exhibited no immunostaining, while 15 specimens from older individuals exhibited only rare immunostaining. Immunostaining did not appear to co-localize selectively with any specific pituitary hormone, although the distribution of immunoreactivity did overlap that of some corticotrophs and was seen in elongated processes of S-100-containing folliculostellate cells. Lipocortin-1 was also found in epithelial cells lining colloid cysts of the residual pars intermedia in 115 of 118 pituitaries ranging in age from 23 weeks' gestation to 83 years. In many intermediate lobe cysts, lipocortin-1 exhibited a pattern of immunoreactivity that partially overlapped the distribution of S-100 protein immunostaining, although the pattern was not identical. Pre-absorption of anti-lipocortin-1 antiserum with lipocortin-1-coupled Sepharose-4B immunoreactivity resulted in loss of immunoreactivity in both lobes. No lipocortin-1 immunoreactivity was seen in the neurohypophysis.

Ontogeny of epidermal growth factor receptor and lipocortin-1 in fetal and neonatal human lungs

The ontogeny and distribution of the epidermal growth factor (EGF) receptor and lipocortin-1, a major cellular substrate of the EGF receptor, were evaluated in a developmental series of fetal and neonatal human lungs (8 to 41 weeks' gestation and stillborn to 16 days' postnatal age). The peroxidase anti-peroxidase technique with two polyclonal antibodies recognizing the EGF receptor and one polyclonal antibody recognizing lipocortin-1 were used for immunohistochemical localization. Extensive or scattered bronchiolar EGF receptor immunoreactivity appeared in the entire series of frozen lung specimens from 15 to 32 weeks' gestation. Bronchial glands exhibited EGF receptor immunostaining from 19 weeks onward, and immunoreactivity in bronchial epithelium was detected from 23 weeks onward. Most tracheas showed extensive lipocortin-1 immunoreactivity in the epithelium beginning at 10 weeks' gestation. Immunostaining was also seen in cells lining the ducts of submucosal glands after 15 weeks' gestation and in nonmucous acinar cells of tracheal glands after their appearance at 18 weeks' gestation. Bronchial epithelium exhibited lipocortin-1 immunoreactivity from 12 weeks' gestation onward. Bronchial gland necks became immunostained from 16 weeks' gestation onward, followed by acinar immunostaining as they subsequently developed. Bronchiolar epithelium was immunostained as early as 12 weeks, beginning with the largest airways, and by 24 weeks extending distally to the bronchioloalveolar portals. Lipocortin-1 immunostaining of larger conducting airway epithelium was primarily confined to ciliated cells. Neither EGF receptor nor lipocortin-1 immunoreactivity was detected in alveolar type I or type II cells, fibrocytes, chondrocytes, or smooth muscle cells at any gestational age. These developmental patterns suggest that the EGF receptor and lipocortin-1 may participate in normal growth factor-induced proliferation of the conducting airways and their glands in the human fetal lung and trachea.

Lipocortin-1 Immunoreactivity in the Normal Human Central Nervous System and Lesions with Astrocytosis

The authors have examined the cellular distribution of lipocortin-1 (L-1) in the normal and diseased central nervous system (CNS) using the peroxidase-antiperoxidase (PAP) technique with a polyclonal antibody specific for L-1. L-1 immunoreactivity was evaluated in the frontal cortex, parahippocampal gyrus/lateral ventricle, cerebellum, medulla, and spinal cord from 27 normal human fetuses, neonates, and adults without neurologic disease and in these same regions and representative lesions from 35 patients with diseases producing varying degrees of astrocytosis, including intraparenchymal hemorrhage; embolic, thrombotic, or traumatic infarctions; and Alzheimer's disease (AD). L-1 immunoreactivity was identified in ependymocytes, choroid plexus epithelia, and scattered subependymal astrocytes throughout the ventricular system from 15 weeks gestation through 82 years of age in both normal and diseased CNSs. L-1 immunoreactivity was also detected in reactive astrocytes and many macrophages surrounding each infarction regardless of site or pathogenesis and in scattered reactive astrocytes in people with AD or SDAT. The limited distribution of L-1 in CNS is consistent with the low amounts of L-1 found in brain and suggests that L-1 may participate in the normal function of ependymocytes and the pathophysiology of reactive astrocytosis.

Lipocortin-1 immunoreactivity in central and peripheral nervous system glial tumors

We examined the cellular distribution of lipocortin-1 (L-1), a major physiologic substrate for the epidermal growth factor receptor/kinase, in 122 central nervous system (CNS) and peripheral nervous system (PNS) neoplasms using the peroxidase-antiperoxidase technique with a polyclonal antibody specific for L-1. Extensive L-1 immunoreactivity was demonstrated in many CNS tumors; in 11 of 21 glioblastoma multiformes, in five of 12 anaplastic astrocytomas, and in five of 14 astrocytomas. Significant numbers of immunoreactive ependymocytes or astrocytes were also seen in six of 13 ependymomas. In contrast, no immunostaining was detected in the oligodendrocytes in any of ten oligodendrogliomas. PNS tumors, found in two of five malignant nerve sheath tumors, 13 of 15 schwannomas, 13 of 17 neurofibromas, and 14 of 15 traumatic neuromas, also contained considerable L-1 immunoreactivity in Schwann cells or mast cells. These findings raise the possibility that L-1 may participate in the proliferation or subsequent differentiation of neoplastic astrocytes, ependymocytes, and Schwann cells.

Ontogeny of Epidermal Growth Factor Receptor/Kinase and of Lipocortin-1 in the Ovine Lung

We have examined the ontogeny and distribution of the epidermal growth factor receptor/kinase (EGF receptor) and of lipocortin-1, a major cellular substrate of the EGF receptor, in a developmental series of 13 normal ovine fetal lungs (44-145 d of gestation) using the peroxidase anti-peroxidase technique with two extensively characterized polyclonal antibodies recognizing the EFG receptor and one polyclonal antibody recognizing lipocortin-1. Immunoreactive EGF receptor/kinase and lipocortin-1 were detected in conducting airway epithelium by the end of the first trimester of pregnancy before bronchial glands could be identified. This was followed at two-thirds of gestation by immunoreactivity in bronchial glands and large bronchioles adjacent to positive bronchi. By seven-eighths of gestation conducting airway epithelium no longer contained consistently detectable immunostaining for EGF receptor, although lipocortin-1 was identified until term in all levels of conducting airways. In contrast, neither EGF receptor nor lipocortin-1 immunoreactivity was detected in alveolar type I or type II epithelial cells, fibrocytes, chondrocytes, smooth muscle, or endothelial cells at any gestational age. These findings suggest that EGF receptor and lipocortin-1 may participate in early airway development.