Lipopolysaccharide induces Rac1-dependent reactive oxygen species formation and coordinates tumor necrosis factor-alpha secretion through IKK regulation of NF-kappa B.

Reactive oxygen species (ROS) are important second messengers generated in response to many types of environmental stress. In this setting, changes in intracellular ROS can activate signal transduction pathways that influence how cells react to their environment. In sepsis, a dynamic proinflammatory cellular response to bacterial toxins (e.g. lipopolysaccharide or LPS) leads to widespread organ damage and death. The present study demonstrates for the first time that the activation of Rac1 (a GTP-binding protein), and the subsequent production of ROS, constitutes a major pathway involved in NFkappaB-mediated tumor necrosis factor-alpha (TNFalpha) secretion following LPS challenge in macrophages. Expression of a dominant negative mutant of Rac1 (N17Rac1) reduced Rac1 activation, ROS formation, NFkappaB activation, and TNFalpha secretion following LPS stimulation. In contrast, expression of a dominant active form of Rac1 (V12Rac1) mimicked these effects in the absence of LPS stimulation. IKKalpha and IKKbeta were both required downstream modulators of LPS-activated Rac1, since the expression of either of the IKK dominant mutants (IKKalphaKM or IKKbetaKA) drastically reduced NFkappaB-dependent TNFalpha secretion. Moreover, studies using CD14 blocking antibodies suggest that Rac1 induces TNFalpha secretion through a pathway independent of CD14. However, a maximum therapeutic inhibition of LPS-induced TNFalpha secretion occurred when both CD14 and Rac1 pathways were inhibited. Our results suggest that targeting both Rac1- and CD14-dependent pathways could be a useful therapeutic strategy for attenuating the proinflammatory cytokine response during the course of sepsis.

Developmental expression of catenins and associated proteins during submucosal gland morphogenesis in the airway.

Although lymphoid enhancer binding factor-1 (Lef-1) plays an obligatory role in airway submucosal gland (SMG) development, its expression alone is not an adequate signal for initiating gland morphogenesis. Because Lef-1 forms a bipartite transcription factor with beta-catenin to mediate wnt pathway signaling, we investigated the expression of beta-catenin and associated proteins during SMG development with both in situ hybridization and immunocytochemistry. Unexpectedly, high levels of E-cadherin mRNA were expressed by cells in developing gland buds from the earliest stages through subsequent differentiation into mature glands. In contrast, a decreased level of E-cadherin immunoreactivity in stage I gland bud cells suggested that post-translational modulation of E-cadherin protein levels may play a critical role in early stages of gland morphogenesis. Adenomatous polyposis coli (APC) mRNA was expressed relatively weakly in the developing ferret trachea, but higher levels of protein staining were observed throughout the cytoplasm of gland buds and surface epithelial cells. B-Catenin mRNA was abundantly expressed throughout the tracheal epithelium and at the highest levels in primordial gland buds. B-Catenin protein localized to the basolateral membranes of all airway epithelial cell types. However, no detectable increases in nuclear or cytoplasmic staining were associated with gland buds, as would be expected if beta-catenin served as a transcriptional cofactor for Lef-1 in gland morphogenesis. Additional studies demonstrated the gamma-catenin distribution to be remarkably similar to that of beta-catenin, whereas alpha-catenin staining was more diffuse in the cytoplasm of airway epithelial and gland bud cells. These descriptive results do not rule out a role for wnt signaling in SMG development , but provide no evidence that beta-catenin, or gamma-catenin, is a cofactor in Lef-1 regulation of SMG development.

Submucosal gland development in the airway is controlled by lymphoid enhancer binding factor 1 (LEF1).

Previous studies have demonstrated that transcription of the lymphoid enhancer binding factor 1 (Lef1) gene is upregulated in submucosal gland progenitor cells just prior to gland bud formation in the developing ferret trachea. In the current report, several animal models were utilized to functionally investigate the role of LEF1 in initiating and supporting gland development in the airway. Studies on Lef1-deficient mice and antisense oligonucleotides in a ferret xenograft model demonstrate that LEF1 is functionally required for submucosal gland formation in the nasal and tracheal mucosa. To determine whether LEF1 expression was sufficient for the induction of airway submucosal glands, two additional model systems were utilized. In the first, recombinant adeno-associated virus was used to overexpress the human LEF1 gene in a human bronchial xenograft model of regenerative gland development in the adult airway. In a second model, the LEF1 gene was ectopically overexpressed under the direction of the proximal airway-specific CC10 promoter in transgenic mice. In both of these models, morphometric analyses revealed no increase in the number or size of airway submucosal glands, indicating that ectopic LEF1 expression alone is insufficient to induce submucosal gland development. In summary, these studies demonstrate that LEF1 expression is required, but in and of itself is insufficient, for the initiation and continued morphogenesis of submucosal glands in the airway.

Spinal cord afferent systems containing the nerve terminal protein NT75.

In the adult spinal cord, immunocytochemical staining for NT75 is concentrated in nerve terminals in the superficial laminae of the dorsal horn. Deeper laminae of the dorsal horn contain moderate immunocytochemical labeling, but the ventral horn is only sparsely stained. The origin of spinal nerve terminals containing NT75 was investigated with lesion techniques, colchicine treatment, and retrograde tracing in combination with immunocytochemical staining. Primary afferent neurons express NT75 immunoreactivity and account for most of the dense staining in the superficial dorsal horn and part of the labeling in the deeper laminae. It was found that corticospinal and virtually all brainstem neurons with descending projections to the spinal cord also express NT75 immunoreactivity, including those terminating in the ventral horn. Colchicine treatment of the spinal cord also resulted in NT75 staining in most, if not all, spinal neurons. It appears that neurons in all three major sources of spinal afferents (primary sensory, descending, and intrinsic systems) can express NT75 immunoreactivity, but that some neurons normally contain higher levels of the protein in their nerve terminals. Previous analysis of developing spinal cord has shown widespread, dense NT75 labeling throughout the spinal gray in the early postnatal period, which later becomes restricted to the adult pattern. These studies support the hypothesis that many spinal pathways express high levels of NT75 immunoreactivity during development, but that only certain pathways maintain high levels in the adult.

Alteration in the pattern of nerve terminal protein immunoreactivity in the perforant pathway in Alzheimer's disease and in rats after entorhinal lesions.

Neurons in layer II of the entorhinal cortex consistently develop neurofibrillary tangles in Alzheimer's disease (AD). Experimental neuroanatomical studies have shown that these neurons give rise to the perforant pathway, a major excitatory projection to the hippocampal formation, which terminates in a discrete pattern in the outer portion of the molecular layer of the dentate gyrus. The distribution of two nerve terminal associated proteins, synaptophysin and NT75, was studied in the molecular layer of the dentate gyrus in AD and control cases to determine whether Alzheimer neuronal pathology is associated with loss of synaptic markers. In parallel studies, the effect of ablation of the entorhinal cortex in rats was evaluated. In AD as compared to controls, a decrease in synaptophysin immunostaining was evident in the terminal zone of the perforant pathway. NT75 nerve terminal immunostaining was too weak to interpret in the human hippocampal formation. Both synaptophysin and NT75 immunoreactivity were found in association with some neuritic plaques. In rats, entorhinal lesions resulted in diminished immunoreactivity for both synaptophysin and NT75 in the perforant pathway terminal zone. These results suggest that nerve terminal protein loss is a concomitant feature of neuronal pathology in AD.

Distribution and developmental expression of the nerve terminal protein NT75 in the rat cerebellum.

Previous studies of the nerve terminal protein NT75 in the developing spinal cord have suggested an association between the appearance of NT75 immunoreactivity and the process of synaptogenesis. To examine the time course of NT75 expression further, the current study compared the localization of NT75 and the synaptic vesicle protein synaptophysin in the adult and developing rat cerebellum and in cerebellar tissue cultures. In the adult cerebellum, dense NT75 staining is confined to the molecular layer, where it is associated with parallel fiber endings of cerebellar granule cells. During development, NT75 immunoreactivity is first detectable in the cerebellar cortex as a dense band of staining in the deepest portion of the molecular layer at postnatal day 10. The stained zone expands to occupy a progressively greater portion of the molecular layer until about postnatal day 20. Synaptophysin staining occurs in granule cell processes earlier than NT75 and is found throughout the molecular layer by postnatal day 7. Quantitatively, rapid increases in both NT75 and synaptophysin occur in the first three postnatal weeks, with NT75 activity reaching levels exceeding the adult value by 50% over postnatal days 20 through 30, whereas synaptophysin plateaus at near adult levels by postnatal day 20. In cerebellar cultures, NT75 staining in neurites develops over several days, increasing coincidentally with development of synaptic contacts, whereas synaptophysin staining is already present in most neurites after only 1 day in vitro. The results indicate that NT75 expression in developing cerebellar granule cell nerve terminals is closely associated with the appearance of mature nerve terminals, suggesting that the protein may have a role in the formation/stabilization of the synaptic ending or in the mechanisms of synaptic transmission.

Immunolocalization and quantitation of a novel nerve terminal protein in spinal cord development.

In the adult spinal cord, the neuron-specific protein NT75 is located in nerve terminals synapsing in the superficial laminae of the dorsal horn. The present study examines the occurrence of NT75 in the developing rat spinal cord. NT75 immunoreactivity is detectable in primary afferent axons at the dorsal root entry zone on embryonic day 15. Subsequently, staining of presumptive nerve terminals appears in the deeper laminae of the dorsal horn, expanding into the superficial laminae during the first postnatal week. NT75 staining also appears in developing corticospinal tract axons in the brainstem at birth, and at lumbosacral levels by postnatal day 5. As NT75-positive nerve terminals approach the adult distribution, staining of primary afferent and corticospinal axons decreases, becoming undetectable by postnatal day 30. Dense transient staining of presumed nerve terminals in the ventral horn is also apparent during early postnatal development. Quantitative analysis of developing spinal cord shows a low level of NT75 immunoreactivity at birth. NT75 activity then increases substantially, reaching values by the third and fourth postnatal weeks up to 2.5 times that seen in adults. The occurrence of NT75 immunoreactivity correlates with the reported time course of synaptic development in the spinal cord. In addition, the results suggest that NT75 immunoreactivity is maintained at high levels in the nerve terminals of certain neural pathways into adulthood, whereas in other systems NT75 immunoreactivity may be detectable only during development.

A nerve terminal protein with a selective distribution in spinal cord and brain.

A monoclonal antibody, designated S-7B8, recognizes a protein antigen localized to highly selected populations of nerve terminals in spinal cord and brain. The antibody produces dense immunocytochemical staining of primary afferent endings that synapse in superficial laminae of the spinal cord dorsal horn. Electron microscopy shows staining to be localized in nerve terminals where reaction product is associated primarily with spherical vesicles. In brain, S-7B8 immunoreactivity occurs in nerve terminals in sensory relay nuclei, most thalamic nuclei, and other selected areas, including the cerebellar molecular layer, the substantia nigra, the globus pallidus, and certain synaptic layers of the hippocampus and dentate gyrus. Endocrine glands and other tissues do not exhibit S-7B8 immunoreactivity. Although the antibody localizes to certain populations of nerve terminals that may use excitatory amino acid neurotransmitters, the distribution of S-7B8 immunoreactivity in the CNS does not correspond to that of any previously identified nerve terminal protein. Experiments to characterize the S-7B8 antigen indicate it may be an integral membrane component since extraction of synaptosomes with alkaline pH or high ionic strength does not release the antigen from the membranes. To identify the molecular weight of the S-7B8 antigen, synaptosomal membranes were solubilized in CHAPS and sequentially chromatographed on hydroxylapatite and then on DEAE anion-exchange resin to produce enriched fractions. When enriched fractions were separated on SDS-PAGE and Western blotted, the S-7B8 antibody specifically stained a protein migrating at 75,000 Da. This protein has been designated NT75. Preliminary studies of developing pathways show that the appearance of S-7B8 immunoreactivity in growing nerve endings corresponds closely to the time when synaptic connections are formed. Thus, the NT75 protein recognized by the S-7B8 antibody may have a role in the development and maintenance of specific synaptic endings.

Intraneuronal IgG in the central nervous system.

The rat central nervous system was examined immunocytochemically for the presence of endogenous IgG. Examination of representative sections of the neuraxis revealed specific staining for IgG in the pia mater and pial vasculature, the ependyma, and diffusely in the hypothalamus and area postrema where the blood-brain barrier is permeable to large molecules. In addition, intraneuronal staining for IgG was noted in specific nuclei including the ventral horn nuclei and intermediolateral nuclei of the spinal cord, the dorsal motor nucleus of the vagus, the nucleus ambiguous, the motor nucleus of the trigeminal, the hypoglossal, facial, and oculomotor nuclei, nuclei projecting to the pituitary and area postrema, and Purkinje cells. The uptake of immunoglobins by these cell groups may have important implications for the pathogenesis of motor and autonomic neuropathies and neuropathies.

Axonal transport of monoclonal antibodies.

Three monoclonal antibodies against rat brain synaptosomes, produced by conventional hybridoma techniques, were screened for their ability to undergo uptake and axonal transport in vivo. Injections of ascitic fluid or of purified immunoglobulin G (IgG) were made into the vitreal chamber of the eye in anesthetized rats to test for anterograde transport in retinal afferents to the contralateral superior colliculus. Retrograde transport by facial nucleus motoneurons was evaluated after injections of antibody into the mystatial vibrissal skin and musculature. Transported immunoglobulins were localized in tissue sections using a modification of the peroxidase-antiperoxidase technique. One monoclonal antibody, S-2C10, was found to undergo anterograde transport in retinal ganglion cells and retrograde axonal transport in facial motoneurons. Transported immunoglobulins were detectable even after injections of dilute antibody solution (0.01-0.05% IgG), and the uptake-transport process for this antibody appeared saturable. Two other antibodies tested, S-4E9 and S-1G10, exhibited the ability to undergo retrograde transport, but only after injections at relatively high antibody concentrations (greater than or equal to 1.0% IgG). Neither of these antibodies was shown to undergo anterograde transport. Following retrograde transport in motoneurons, the S-2C10 antibody was localized in neuronal perikarya, proximal dendrites, and the adjacent neuropil of the facial motor nucleus. In contrast, the S-4E9 and S-1G10 antibodies were localized in punctate granules within neuronal cell somata following transport. The findings suggest that the uptake-transport process for the S-2C10 antibody is mediated by adsorptive endocytosis following binding of the antibody to a plasma membrane component (or components) present in somadendritic and nerve terminal membranes.(ABSTRACT TRUNCATED AT 250 WORDS)

Axonal transport of antibodies to subcellular and protein fractions of rat brain.

Experiments examined the feasibility of using the axonal transport of antibodies as a possible means to characterize nerve membrane composition and the fate of internalized macromolecules. Polyspecific antibodies were generated in rabbits against rat brain synaptosomal and microsomal subcellular fractions and against wheat germ agglutinin-binding proteins isolated by lectin affinity chromatography. Antisera were injected into the vitreal chamber of the eye and into the facial musculature of anesthetized rats to test, respectively, for anterograde transport in retinotectal neurons and for retrograde transport in facial motoneurons. Control injections of preimmune serum were made into the opposite side. After survival for 4-168 h, animals were perfused and the axonally transported rabbit immunoglobulins detected in frozen sections of the brainstem using a modified peroxidase-antiperoxidase immunocytochemical procedure. Antisera against all 3 classes of neuronal antigens contained antibodies that underwent retrograde axonal transport. No evidence of anterograde transport was seen. Neurons containing retrogradely transported immunoglobulins exhibited punctate as well as diffuse staining of the cytoplasm and proximal dendrites, exclusive of the nucleus. Following retrograde transport of antibodies to the synaptosomal fraction, staining of the neuropil around motoneurons was also observed, suggesting transcellular transport of these antibodies. Concentrations of injected antibodies as low as 1% of whole antiserum led to detectable retrograde transport. Increasing concentrations of antibodies above the amount in whole antiserum did not increase the intensity of staining in retrogradely labeled neurons, suggesting saturation. The findings support the view that antibodies to neural membranes are taken up and transported by binding to specific sites on nerve terminals.(ABSTRACT TRUNCATED AT 250 WORDS)

The central distribution of vagal catecholaminergic neurons which project into the abdomen in the rat.

A double labeling technique employing retrograde labeling of vagal neurons with horseradish peroxidase from injections into the stomach wall and immunocytochemistry for dopamine-beta-hydroxylase revealed catecholaminergic neurons in the medulla oblongata which project into the abdomen. The great majority of such neurons were located in the dorsal motor nucleus of the vagus, particularly in its rostral third.

The descending and intrinsic serotoninergic innervation of an elasmobranch spinal cord.

The descending and the intrinsic components of the serotoninergic (5HT) innervation of the Atlantic stingray spinal cord were described by comparing the distributions of neuronal elements exhibiting 5HT-like immunoreactivity (peroxidase-antiperoxidase method) in sections caudal and rostral to spinal transections. The cells of origin of the descending 5HT system were located with a double labeling method for both retrogradely transported horseradish peroxidase (HRP) and 5HT staining. The descending system provides virtually the entire 5HT innervation of the dorsal horn, the intermediate zone, and the dorsal and lateral portions of the ventral horn. Fibers of the descending 5HT system course in the lateral funiculus, the dorsal portion of the ventral funiculus, and in the submeningeal zones of the dorsal and lateral aspects of the spinal cord. This projection primarily originates from the 5HT cell groups of the caudal rhombencephalon (groups II and III; Ritchie et al., '83), with a minor contribution from group IV in the rostral rhombencephalon. The organization of the descending 5HT system in stingrays is remarkably similar to that of mammals. The intrinsic spinal 5HT system consists of cells distributed in the ventromedial spinal cord that have processes extending longitudinally in a ventral submeningeal fiber network. Fibers were traced from the submeningeal system to the ventral horn, where varicose processes were restricted largely to the neuropil ventral to the somata of the fin motoneurons. The existence of a well-defined intrinsic 5HT system in stingrays supports the hypothesis that such a system exists in the spinal cords of a variety of vertebrates.

The distribution of serotonin in the CNS of an elasmobranch fish: immunocytochemical and biochemical studies in the Atlantic stingray, Dasyatis sabina.

The distribution of serotonin (5HT) in the brain of the Atlantic stingray was studied with peroxidase-antiperoxidase immunocytochemistry and high-pressure liquid chromatography. The regional concentrations of 5HT determined for this stingray fell within the range of values previously reported for fishes. A consistent trend in vertebrates for the hypothalamus and midbrain to have the highest concentrations and the cerebellum the lowest was confirmed in stingrays. Neuronal cell bodies and processes exhibiting 5HT-like immunoreactivity were distributed in variable densities throughout the neuraxis. Ten groups of 5HT cells were described: (I) spinal cord, (II-IV) rhombencephalon, (V, VI) mesencephalon, (VII, VIII) prosencephalon, (IX) pituitary, and (X) retina. There were three noteworthy features of the 5HT system in the Atlantic stingray: (1) 5HT cells were demonstrated in virtually every location in which 5HT-containing cells have been described or alluded to in the previous literature. The demonstration of immunopositive cells in the spinal cord, the retina, and the pars distalis of the pituitary suggests that 5HT may be an intrinsic neurotransmitter (or hormone) in these regions. (2) The distribution of 5HT cells in the brainstem shared many similarities with that in other vertebrates. However, there were many 5HT cells outside of the raphe nuclei, in the lateral tegmentum. It appears that the hypothesis that "lateralization" of the 5HT system is an advanced evolutionary trend cannot be supported. (3) 5HT fibers and terminals were more widely distributed in the Atlantic stingray brain than has been reported for other nonmammalian vertebrates on the basis of histofluorescence. It appears that this feature of the 5HT system arose early in phylogeny, and that the use of immunohistochemistry might reveal a more general occurrence of widespread 5HT fibers and terminals.

Immunocytochemical demonstration of serotonergic neurons and processes in the retina and optic nerve of the stingray, Dasyatis sabina.

Neurons and processes in the stingray retina can be stained using PAP immunohistochemistry and an antibody to serotonin, without pharmacological pretreatment. Most of the cell bodies are in the inner nuclear layer while the processes ramify in the inner plexiform layer suggesting the presence of a population of serotonin containing amacrine cells in this species. Scattered immunopositive axons were observed in the optic nerve from the optic chiasm to the optic nerve head.

Immunohistochemical studies on the distribution and origin of candidate peptidergic primary afferent neurotransmitters in the spinal cord of an elasmobranch fish, the Atlantic stingray (Dasyatis sabina).

The distribution and origin of four peptide neurotransmitter candidates of primary afferents (substance P, SP; somatostatin, SS; cholecystokinin, CCK; and vasoactive intestinal polypeptide, VIP) were studied in the stingray with peroxidase-antiperoxidase (PAP) immunohistochemistry. This elasmobranch has virtually no unmyelinated primary afferents, having instead only large and small myelinated afferents. SP-like immunoreactivity was distributed densely in the superficial aspect of the substantia gelatinosa (SG), particularly laterally, and was scattered in the nucleus proprius, the intermediate zone, and the ventral horn. The distributions of SS-, CCK-, and VIP-like immunoreactivities were similar to each other, but different from that of SP. Stained fibers appeared to issue from a prominent tract in the dorsolateral funiculus to form a plexus at the lateral margin of the nucleus proprius. The fibers spread dorsally and medially through the SG to terminate in a thin band at the superficial margin of the SG. Both SS and CCK were more dense in the lateral third of the SG, while VIP was more diffusely distributed within this structure. The remaining regions of the spinal gray matter contained immunoreactive fibers and terminals in variable densities. Many SS-positive cell bodies were observed in the ventral horn, in the deep dorsal horn, and in the ependymal layer. CCK-positive cells were observed in the medial ventral horn, and VIP-positive cells were observed subjacent to the SG and within the dorsolateral funiculus. After unilateral dorsal rhizotomies, SP-like immunoreactivity in the SG was depleted, while SP staining elsewhere and all SS, CCK, and VIP staining was indistinguishable from control. Thus all four peptides are present in the stingray spinal cord, although only SP appears to be a candidate primary afferent transmitter.

The relationship of the medullary catecholamine containing neurones to the vagal motor nuclei.

We have re-examined in the rat the nuclear localization of the medullary catecholamine-containing cell groups (A1 and A2) and their relation to the vagal motor nuclei using a double labeling method. The vagal nuclei were defined by the retrograde transport of horseradish peroxidase applied to the cervical vagus, and noradrenergic and adrenergic neurons were stained with the peroxidase-antiperoxidase immunocytochemical method using an antibody to dopamine beta-hydrolase. The method allows visualization of both labels within single neurons. The neurons of the A2 group are primarily distributed in both the nucleus of the solitary tract and the dorsal motor nucleus of the vagus in a complex interrelationship that depends on the rostrocaudal level. Caudal to the obex, cells of the dorsal motor nucleus of the vagus are scattered among cells immunoreactive for dopamine beta-hydroxylase in the area considered to be the commissural subnucleus of the nucleus of the solitary tract. At levels near and slightly rostral to the obex, the dopamine beta-hydroxylase-positive cells are largely confined to nucleus of the solitary tract. However, the rostral third of the A2 group lies predominantly within dorsal motor nucleus, as defined by horseradish peroxidase labeled cells, with only a few cells in the nucleus of the solitary tract. A subset of the dopamine beta-hydroxylase positive cells within the rostral dorsal motor nucleus of the vagus are also vagal efferents. Our results suggest that a second population of dopamine beta-hydroxylase positive vagal efferents may exist ventrolaterally where neurons of the AI cell group intermingle with those of nucleus ambiguus.

Immunocytochemical demonstration of serotonergic cells, terminals and axons in the spinal cord of the stingray, Dasyatis sabina.

Serotonin-like immunoreactivity, as demonstrated by the PAP method, was contained within cells as well as fibers and terminals, in the spinal cord of the stingray. Serotonergic spinal neurons were seen on 43% of the sections examined and were restricted to the ventral white matter. Immunoreactive terminals and varicosities were densely distributed over the spinal gray matter at all segmental levels, and stained fibers were seen in all portions of the white matter with the exception of the medial anterior and dorsal funiculi.