Neuroimmunotoxicology: humoral assessment of neurotoxicity and autoimmune mechanisms.

The interactions between the nervous and immune systems have been recognized in the development of neurodegenerative disease. This can be exploited through detection of the immune response to autoantigens in assessing the neurotoxicity of environmental chemicals. To test this hypothesis, the following questions were addressed. a) Are autoantibodies to nervous system (NS) antigens detected in populations exposed to environmental or occupational chemicals? In sera of male workers exposed to lead or mercury, autoantibodies, primarily IgG, to neuronal cytoskeletal proteins, neurofilaments (NFs), and myelin basic protein (MBP) were prevalent. These findings were confirmed in mice and rats exposed to either metal. b) Do autoantibodies to NS antigens relate to indices of exposure? In humans exposed to either metal, and similarly in exposed rats, titers of IgG against NFs and MBP significantly correlated with blood lead or urinary mercury, the typical indices of exposure. c) Do autoantibodies correlate with sensorimotor deficits? In workers exposed to lead or mercury, a significant correlation was observed between IgG titers and subclinical deficits. Doses of metals used in rat exposures were subclinical, suggesting that autoantibodies may be predictive of neurotoxicity. d) Is the detection indicative of nervous system pathology? In rats exposed to metals, histopathology indicated central nervous system (CNS) and peripheral nervous system (PNS) damage. In addition there was evidence of astrogliosis, which is indicative of neuronal damage in the CNS, and the presence of IgG concentrated along the blood-brain barrier, as indicated by immunostaining for antibodies. e) Are immune responses to NS antigens pathogenic? Immunoglobulin fractions from rat and human sera interfered with neuromuscular function. These studies suggest that the detection of autoantibodies to NS-specific antigens may be used to monitor the development of neurotoxicity to environmental chemicals and that immune mechanisms may be involved in the progression of neurodegeneration.

Organophosphate neuropathy due to methamidophos: biochemical and neurophysiological markers.

Neuropathy target esterase (NTE), the putative target enzyme for organophosphate induced delayed polyneuropathy (OPIDP), can be measured in lymphocytes but has rarely been assessed in acute human poisoning. Serum autoantibodies to nervous system proteins develop in hens poisoned with neuropathic insecticides and also have not been studied after human poisoning. Serial lymphocyte NTE (LNTE) was measured in a 16-year-old boy after acute poisoning with methamidophos for evaluation as a predictor of subsequent neuropathy. The profiles of serum autoantibodies to neurofilament triplet proteins, myelin basic protein, and glial fibrillary acidic protein were measured in order to characterize changes occurring as a result of OPIDP. Clinical neuropathy characterized by steppage gate and profound lower extremity weakness, decreased grip and pinch strength, and decreased ulnar and absent tibial compound muscle action potentials developed 2 weeks following poisoning. Sensory examination and nerve conduction studies were normal. On day 3 following poisoning LNTE was depressed (77% compared with subsequent baseline enzyme activity). Marked increases in serum immunoglobulin G (IgG) autoantibodies to glial fibrillary acidic protein and to neurofilament 200 were observed after the development of OPIDP. We conclude that inhibition of lymphocyte NTE is predictive of subsequent OPIDP. Serum autoantibody titers to nervous system proteins may be useful markers of neuropathy.

Autoantibodies to neurofilaments (NF), glial fibrillary acidic protein (GFAP) and myelin basic protein (MBP) in workers exposed to lead.

An expanded follow-up assessment of the autoantibody response to neuronal and astroglial autoantigens (NF68; NF160; NF200; MBP; GFAP) as early markers of neurotoxicity was performed in male workers exposed to lead (Pb) of a battery factory (n=50) and a matched reference group (R) of workers at a food packing plant (n=39). Mean age, years of exposure and blood lead (PbB ug/dl) (+/-SD) for lead and R, respectively, were: Age:39 +/- 6; 41 +/- 7; Yrs. Exposed: 14 +/- 6:0; PbB: 32 +/- 11: 16 +/- 5 percent with detectable titers to nervous system proteins in the Pb and R populations, respectively, were: Anti-NF68: 59; 17; Anti-NF160: 28; 15; Anti-NF200: 25; 0; Anti-GFAP: 90; 20; Anti-MBP: 16; 4. Autoantibodies to nervous system proteins predominated in workers occupationally exposed to Pb compared to R. Anti-NF68 and GFAP titers were the most frequently encountered. Anti-NF68 titers were significantly correlated with years of exposure (r = 0.538, p < 0.0001) and with PbB (r=0.325, p < 0.05). Furthermore, the number of detectable autoantibody types correlated with clinical scores of sensorimotor deficits (r = 0.459, p < 0.0001). This study suggests that autoantibodies provide a promising biomarker of neurotoxicity while providing information on subcellular targets. It also raises concerns of toxicant-induced autoimmune neuropathy.

Ozone-induced alteration in beta-adrenergic pharmacological modulation of pulmonary macrophages.

Ozone is a ubiquitous air pollutant which can affect numerous function s of the respiratory system. However, previous work has not provided any information concerning its ability to modulate pharmacological receptors of pulmonary macrophages. This study examined, using a chemiluminescence assay, the beta-adrenergic modulation of pulmonary macrophages harvested from rabbits exposed for 3 hr/day for 5 days to 0.1, 0.3 or 0.6 ppm ozone (O3) or to 3 hr/day for 20 days to 0.1 or 0.3 ppm. Receptor activity was monitored using release of reactive oxygen species (ROS) following administration to the cells of the beta2-receptor agonist, isoproterenol. An O3-exposure concentration-dependent response was observed for isoproterenol efficacy following 5-day exposures, in that 0.1 ppm O3 induced a significant enhancement of beta-adrenergic inhibition of ROS production, 0.3 ppm ozone produced no significant change from control, and 0.6 ppm decreased inhibition. No significant effects on beta-adrenergic modulation were noted following the 20-day exposures. The results of this study suggest that short-term repeated exposures to O3 are capable of inducing alterations in the pharmacological functioning of pulmonary macrophages, while longer term exposures may result in adaptation. Alterations in receptor function have implications in terms of pulmonary defense and disease.

Exposure to methylmercury results in serum autoantibodies to neurotypic and gliotypic proteins.

Environmental exposure to methylmercury (MeHg) continues to pose a threat to humans, making early detection of neurotoxic effects a pressing concern. An enzyme-linked immunosorbent assay (ELISA) to measure serum autoantibodies (lg) to neurotypic and gliotypic proteins [neurofilament triplet (NF68; NF160; NF200), myelin basic protein (MBP) and glial fibrillary acid protein (GFAP)] as markers of subclinical neurotoxicity was developed and tested in Fisher 344 rats exposed orally to 16 or 32 ppm MeHg. Both levels of MeHg resulted in serum lg to all 5 proteins, not normally seen in controls. For anti-NFs and anti-GFAP, lgM isotype predominated significantly (p < 0.05) over lgG. lg for MBP were of the lgG isotype, lgM were not detected. Significant differences (p < 0.05) between 16 and 32 ppm MeHg in levels of anti-NF 68 and GFAP, lgM, were evident at 7 days of exposure, but not at 14 days. Anti-NF 160, lgM, was significantly (p < 0.01) elevated in rats exposed to 32 ppm vs 16 ppm at 14 days. However, at both dose levels anti-NF 68 titers were the most elevated of the three NF proteins (p < 0.0001). For anti-NF 200 and anti-MBP it was the lgG isotype that was significantly (p < 0.01) elevated in the 32 ppm group at 7 days. GFAP levels as a marker of neurotoxicity were determined in the cortex, hippocampus and cerebellum. Exposure to 32ppm MeHg resulted in decreased (p < 0.05) levels in the cortex at 14 days. Both levels of MeHg resulted in increased GFAP in the cerebellum at 14 days. This study suggests that assay of autoantibodies against nervous system proteins may provide a means of assessing the early neurotoxic effects of environmental MeHg exposure.

Exposure to methyl mercury results in serum autoantibodies to neurotypic and gliotypic proteins.

Environmental exposure to methyl mercury (MeHg) continues to pose a threat to humans, making early detection of neurotoxic effects a pressing concern. An enzyme-linked immunosorbent assay (ELISA) to measure serum autoantibodies (Ig) to neurotypic and gliotypic proteins [neurofilament triplet (NF68; NF160; NF200), myelin basic protein (MBP) and glial fibrillary acid protein (GFAP)] as markers of subclinical neurotoxicity was developed and tested in Fisher 344 rats exposed orally to 16 or 32 ppm MeHg. Both levels of MeHg resulted in serum Ig to all 5 proteins, not normally seen in controls. For anti-NFs and anti-GFAP, IgM isotype predominated significantly (p < 0.05) over IgG.Ig for MBP were of the IgG isotype, IgM were not detected. Significant differences (p < 0.05) between 16 and 32 ppm MeHg in levels of anti-NF 68 and GFAP, IgM, were evident at 7 days of exposure, but not at 14 days. Anti-NF 160, IgM, was significantly (p < 0.01) elevated in rats exposed to 32 ppm vs 16 ppm at 14 days. However, at both dose levels anti-NF 68 titers were the most elevated of the three NF proteins (p < 0.0001). For anti-NF 200 and anti-MBP it was the IgG isotype that was significantly (p < 0.01) elevated in the 32 ppm group at 7 days. GFAP levels as a marker of neurotoxicity were determined in the cortex, hippocampus and cerebellum. Exposure to 32ppm MeHg resulted in decreased (p < 0.05) levels in the cortex at 14 days. Both levels of MeHg resulted in increased GFAP in the cerebellum at 14 days. This study suggests that assay of autoantibodies against nervous system proteins may provide a means of assessing the early neurotoxic effects of environmental MeHg exposure.

Airway smooth muscle relaxant effects of the cocaine pyrolysis product, methylecgonidine.

Methylecgonidine (anhydroecgonine methylester; MEG) is produced when cocaine base ("crack") is heated. Since crack smoking can produce significant airway toxicity and the role of MEG in this toxicity is unknown, we determined the effects of MEG on guinea pig isolated tracheal rings. Trachea do not contract in response to MEG; rather, MEG (10(-9) to 10(-3) M) dose-dependently relaxed tissue precontracted with 2 x 10(-3) M acetylcholine (ACh). MEG (10(-9) to 10(-6) M) reduced the magnitude of contractions induced by ACh, carbachol, histamine and KCl in a nonsurmountable manner; the maximal response to these agents was not restored after repeated washing. MEG did not affect contractions induced by BaCl2. 4-Diphenyl acetoxymethyl piperidine methiodide (4-DAMP; 10(-7) M), in the presence or absence of MEG (10(-7) M), shifted the dose-effect curve for ACh 30-fold to the right. After washing, sensitivity to ACh was fully recovered in tissues exposed to 4-DAMP alone, but was still reduced to 50% of control in tissues exposed to 4-DAMP and MEG. The effects of MEG were unlike those of cocaine which, at 10(-7) to 10(-5) M, increased the magnitude of contractions induced by ACh (10(-9) to 2 x 10(-3) M); MEG (10(-7) M) abolished this increase. The mechanism by which MEG relaxes tracheal smooth muscle has not been established, but it is likely to be independent of direct interaction with sites that mediate the effects of the bronchoconstrictor agents used in this study.(ABSTRACT TRUNCATED AT 250 WORDS)

Nonspecific bronchial responsiveness assessed in vitro following acute inhalation exposure to ozone and ozone/sulfuric acid mixtures.

Air pollution may play some role in the recent increase in severity and prevalence of asthma, but the specific chemical components with the ambient pollutant mix that may be responsible have not been delineated. Since ambient exposures involve mixtures, it is essential to examine airway responses to realistic pollutant mixtures. This study examined the ability of single (3-h) inhalation exposures to ozone and to mixtures of ozone plus sulfuric acid to induce nonspecific airway hyperresponsiveness in healthy rabbits. Airway responsiveness was assessed using an in vitro assay involving administration of increasing doses of acetylcholine to bronchial rings obtained from animals exposed to 0.1-0.6 ppm ozone or to mixtures of ozone and 50-125 micrograms/m3 sulfuric acid aerosol; results were compared to those reported previously for sulfuric acid alone. Bronchial hyperresponsiveness to ozone was noted following exposure at all concentrations, but the combination of pollutants results in antagonism. The results support the potential for ozone to induce airway hyperresponsiveness in healthy animals and suggest that interaction with sulfuric acid may reduce the effectiveness of both pollutants.

Lead alters the immunogenicity of two neural proteins: a potential mechanism for the progression of lead-induced neurotoxicity.

Some heavy metals have been suspected of playing a role in the pathogenesis of nervous system diseases such as multiple sclerosis, amyotrophic lateral sclerosis, and Alzheimer's disease. In these disorders, autoantibodies against neural proteins are evident at some stage of the disease. Lead is known to affect both the immune and nervous systems. Work in our laboratory has shown that lead exposure leads to the production of autoantibodies against neural proteins, including myelin basic protein (MBP) and glial fibrillary acidic protein (GFAP). We hypothesize that lead aggravates neurological disease by enhancing the immunogenicity of nervous system proteins, including MBP and GFAP. To test this hypothesis, lead-altered protein was prepared by incubating MBP or GFAP with lead acetate for 24 hr. On days 0, 14, and 28, mice received inoculations with either saline, native protein, or lead-altered protein. Anti-MBP and anti-GFAP, isotypes IgM and IgG, were measured in sera by ELISA on day 38. Sera of mice treated with lead-altered MBP had statistically higher anti-MBP IgG titers than both control and native MBP-immunized mice. An analogous response was seen in mice immunized with lead-altered GFAP. Supernatants from lectin-stimulated splenocytes were also examined for antibody titers and for interleukin 2 (IL-2) and interleukin 6 (IL-6) levels. A significant increase in IL-6 production was seen in mice immunized with lead-altered MBP but not with lead-altered GFAP. No changes were observed in the IL-2 levels of mice immunized with either lead-altered protein.(ABSTRACT TRUNCATED AT 250 WORDS)

Nonspecific airway hyperresponsiveness induced by inhalation exposure to sulfuric acid aerosol: an in vitro assessment.

Air pollution may be a factor in the recent increase in severity and prevalence of asthma, but the specific chemical components within the ambient pollutant mix which may be responsible have not been delineated. Acid sulfate aerosols, such as sulfuric acid, have been associated with exacerbation of symptoms in asthmatics and induction of nonspecific airway hyperresponsiveness in normal laboratory animals. This study examined the ability of single (3 hr) inhalation exposures to sulfuric acid to induce nonspecific airway hyperresponsiveness in healthy rabbits. Responsiveness was assessed using an in vitro assay involving administration of increasing doses of acetylcholine or histamine to bronchial and tracheal rings obtained from animals exposed to 50-500 micrograms/m3 sulfuric acid aerosol. Bronchial hyperresponsiveness to both agonists was noted following exposures at > or = 75 micrograms/m3. In addition, sulfuric acid altered the normal response pattern of trachea to histamine. The results provide further support for a role of acid sulfates in the induction of airway hyperresponsiveness, and suggest that effects may be due to modulation of pharmacological receptors involved in maintaining airway tone.

Comparison of organophosphate-induced delayed neuropathy between branches of the tibial nerve and the biventer cervicis nerve in chickens.

Delayed neuropathy induced by organophosphorus esters has been reported to be more selective for large diameter myelinated fibers, especially in distal portions of long nerves. This concept was re-evaluated in chickens by quantitatively comparing the effects of the organophosphates tri-ortho-tolyl (TOTP) and phenyl saligenin phosphate (PSP) on two separate nerves, the branch of the tibial nerve that supplies the gastrocnemius muscle, and the small cervical nerve that innervates the biventer cervicis muscle. Histograms illustrating the distribution of myelinated fibers within each nerve showed that the biventer nerve is composed of a population of fibers smaller than those within the tibial nerve branch. However, the number of myelinated fibers measured per mm2 of endoneurium was reduced in both nerves 10 and 15 days after organophosphate administration, providing indirect evidence that fiber diameter is not critical in determining susceptibility to organophosphorus-induced delayed neuropathy (OPIDN). More direct evidence was provided by fiber diameter histograms of both biventer nerves and tibial nerve branches taken from hens that received PSP. In comparison to control values, there was a decrease in all fiber sizes in both nerves, indicating that factors other than axonal size are important in determining nerve fiber susceptibility to OPIDN.

Use of the biventer cervicis nerve-muscle preparation to detect early changes following exposure to organophosphates inducing delayed neuropathy.

Indices of organophosphorus (OP)-induced delayed neuropathy (OPIDN) in the hen model have traditionally been restricted to the early inhibition of neuropathy target esterase (NTE) and ataxia with associated pathological changes in hind limb peripheral nerve which occur more than 7 days after OP exposure. The biventer cervicis nerve-muscle preparation was used to evaluate OPIDN in adult hens at various time periods after treatment with either the protoxicant tri-o-tolyl phosphate (TOTP), 360 mg/kg po, or the active congener phenyl saligenin phosphate (PSP), 2.5 mg/kg im. NTE activity was 21 and 48% of control for TOTP and PSP, respectively, 4 days after administration. Clinical signs were notable by 10 days and progressed in severity to paralysis by 21 days. Partial clinical recovery was evident at 37 days. Denervation hypersensitivity of biventer cervicis muscle to acetylcholine (ACh) was evident as early as 4 days following TOTP or PSP treatment. The sensitivity to ACh was greatest 21 days after OP administration, with partial recovery at 37 days. Strength-duration curves (SDC) of preparations from OP-treated hens showed an increase in excitability thresholds and elevated rheobase with shorter chronaxie than did preparations from controls as early as 4 days following treatment with either compound. SDC at 37 days indicated partial reinnervation. Peripheral nerve myelinated fiber degeneration and regeneration consistent with these physiological changes was seen on histopathological examination. This study suggests that the biventer cervicis nerve-muscle preparation may prove useful for detection of functional and morphological changes that occur during the interval between NTE inhibition and appearance of clinical deficits.

Protease activity in brain, nerve, and muscle of hens given neuropathy-inducing organophosphates and a calcium channel blocker.

Activity of calcium-activated neutral protease (CANP or calpain), an enzyme responsible for degradation of axonal and muscle cytoskeletal elements, was determined in brain, sciatic nerve, and gastrocnemius muscle of hens given tri-ortho-tolyl phosphate (TOTP, 360 mg/kg po) or active congener phenyl saligenin phosphate (PSP, 2.5 mg/kg im) with and without a calcium channel blocker which ameliorated clinical signs of organophosphate-induced delayed neuropathy (nifedipine 1 mg/kg/day x 5). Calcium channel blocker administration was initiated 1 day prior to administration of organophosphate (OP). OP administration caused an increase in CANP activity in brain within 4 days and in sciatic nerve and gastrocnemius muscle within 2 days of administration. This increase did not occur if nifedipine was administered to PSP-treated hens. Total sciatic nerve calcium concentrations were also increased by PSP, but not until OP-treated hens were no longer being administered calcium blockers. This indicates that calcium channel blockers may contribute to amelioration of organophosphate-induced delayed neuropathy by attenuation of calcium-mediated disruption of axonal and muscle cytoskeletal homeostasis.

Modification of phenyl saligenin phosphate-induced delayed effects by calcium channel blockers: in vivo and in vitro electrophysiological assessment.

Effects of organophosphorus esters (OPs) inducing delayed neuropathy in the adult hen have traditionally been evaluated by assessment of morphology and function of nerve and muscle in the rear limbs of animals exposed. In this study, organophosphorus-induced delayed neuropathy (OPIDN), including neuromuscular function and histology, were studied in vivo using sciatic nerve, tibial nerve and gastrocnemius muscle in anesthetized hens that had been administered phenyl saligenin phosphate (PSP), 2.5 mg/kg by intramuscular injection. In addition, OPIDN was examined in vitro using the biventer cervicis nerve and muscle of the same adult hens. Both nerve-muscle preparations were used for construction of strength duration curves (SDC) on days 4-5, 7-8, and 15-16 after PSP; the biventer cervicis preparation was also used 21-22, 37 and 64 days after PSP administration. Histological examination was done at these same time periods. SDC revealed significant increases in excitability thresholds for preparations from hens receiving PSP only compared to preparations from control hens, or compared to preparations from hens treated with PSP and either nifedipine (1 mg/kg intramuscularly for 5 days), or verapamil (7 mg/kg intramuscularly for 4 days), with treatment beginning 24 hours before administration of PSP. Ataxia, which appeared 7-10 days after hens were given PSP, was less pronounced in hens given PSP plus either calcium channel blocker than in hens given PSP alone. Whether treatment was initiated before or after PSP, verapamil, a phenylalkylamine, reduced sensitivity of the biventer cervicis muscle to acetylcholine-induced stimulation. The dihydropyridine, nifedipine, was less effective at reducing muscle sensitivity to acetylcholine post-exposure than when used as a pretreatment. Lesions were extensive in the biventer cervicis nerve after PSP administration and modification by treatment with calcium channel blockers was evident.

Effect of verapamil on organophosphorus-induced delayed neuropathy in hens.

Verapamil, a calcium channel blocker, was administered to adult white leghorn hens to determine if inhibition of calcium entry could alter delayed neuropathy induced by administration of phenyl saligenin phosphate (PSP). Verapamil was given im in doses of 7 mg/kg/day for 4 days beginning 24 hr before administration of PSP (2.5 mg/kg im). Ataxia was less pronounced in hens given PSP plus verapamil than in hens given PSP alone during observations made 8-28 days after PSP administration. Myelinated fiber lesions were less extensive and regeneration more notable in the biventer cervicis nerve in chickens given PSP plus verapamil, with samples obtained both 17 and 28 days after PSP. In the absence of verapamil, rheobase and chronaxie values of strength-duration curves were higher and shorter, respectively, and sensitivity to acetylcholine was increased in biventer cervicis nerve-muscle preparations from hens given PSP. Verapamil did not alter PSP-induced inhibition of neurotoxic esterase, indicating that the mechanism involved in amelioration of these indices of delayed neuropathy was not associated with initial enzyme inhibition caused by this organophosphorus ester.

The biventer cervicis nerve-muscle preparation of adult hens: effects of phenyl saligenin phosphate administration.

A biventer cervicis nerve-muscle preparation was used to assess in vitro neuromuscular function in adult white leghorn hens with clinical signs of delayed neuropathy induced by phenyl saligenin phosphate (PSP). Denervation of fast-twitch muscle fibers 13-15 days after PSP was indicated by higher excitability thresholds and by discontinuities of the strength-duration curves. Nerve degeneration was also indicated by significantly elevated rheobase values for all three experimental groups (2, 6 and 10 mg/kg PSP, im) and by shorter chronaxie for preparations from hens receiving 6 and 10 mg/kg. Chronaxie values for preparations from hens given 2 mg/kg PSP were longer than controls, indicating only partial denervation. Biventer cervicis muscle from all PSP-treated hens was 100-1000x more sensitive to acetylcholine (ACh) than muscle from untreated hens, a response typical of denervated slow-tonic muscle. Tension development in response to ACh was 20-45x greater than control in muscle of PSP-treated hens. The greatest sensitivity and tension development in response to ACh was encountered in muscles from hens given 10 mg/kg PSP. Denervation was also indicated histologically by the extensive degeneration and loss of larger myelinated nerve fibers. This study indicates that alteration in neuromuscular function and morphology occurs in the neck region of chickens during OPIDN and that deficits in nerves innervating both fast-twitch and slow-tonic muscles can be differentiated by nerve stimulation and by denervation hypersensitivity to ACh.