|Year : 2014 | Volume
| Issue : 3 | Page : 198-204
Anti-nuclear antibodies positive serum from systemic lupus erythematosus patients promotes cardiovascular manifestations and the presence of human antibody in the brain
Marie Kelly-Worden, Leslie Hammer, Robyn Gebhard, Lauran Schrader, Marley Griffin, Dalahnna Cooper
Department of Physiology and Health Science, Ball State University, Muncie, IN 47306, USA
|Date of Submission||30-Oct-2013|
|Date of Decision||22-Jan-2014|
|Date of Acceptance||22-Jan-2014|
|Date of Web Publication||24-Jun-2014|
Department of Physiology and Health Science, Ball State University, Muncie, IN 47306
Source of Support: This project was funded through Ball State University
ASPiRE grants, the Ball State Chapter of Sigma Xi and our laboratory’s
Neuroscience Research account, Conflict of Interest: None
| Abstract|| |
Background: Systemic lupus erythematosus (SLE) is characterized by the presence of anti-nuclear antibodies (ANAs) in the serum of patients. These antibodies may cross over into the brain resulting in the development of neuropsychiatric symptoms and result in abnormal pathology in other organs such as the heart and kidneys. Objective: The objective of this study was to determine if SLE pathology could be detected in the hearts and brains of rats injected with positive human ANA serum. Materials and Methods: Lewis rats (n = 31) were selected for this study due to documented research already performed with this strain in the investigation of serum sickness, encephalitis and autoimmune related carditis. Rats were injected once a week with either ANA positive or negative control serum or saline. Hearts were examined for initial signs of heart disease including the presence of lipid deposits, vegetation, increased ventricular thickness and a change in heart weight. Brains were examined for the presence of human antibody and necrotic lesions. Animals were observed for outward signs of neuropathy as well. Blood samples were taken in order to determine final circulating concentrations of IgG and monitor histamine levels. Results: Animals injected with ANA were significantly higher for lipid deposits in the heart and an increased ventricular thickness was noted. One animal even displayed Libman-Sacks endocarditis. Brains were positive for the presence of human IgG and diffuse internal lesions occurred in 80% of the ANA positive serum injected animals examined. Blood histamine levels were not significantly different, but actually lower than controls by the end of the experiment. Conclusion: Since human antibodies were detected in the brain, further studies will have to identify which antibody cross reactions are occurring within the brain, examine cell infiltration as well as characterize the antibodies associated with more destructive consequences such as lesion formation.
Keywords: Anti-nuclear antibodies, brain, heart, histamine, Libman-Sacks endocarditis, rat, systemic lupus erythematosus
|How to cite this article:|
Kelly-Worden M, Hammer L, Gebhard R, Schrader L, Griffin M, Cooper D. Anti-nuclear antibodies positive serum from systemic lupus erythematosus patients promotes cardiovascular manifestations and the presence of human antibody in the brain. J Pharm Bioall Sci 2014;6:198-204
|How to cite this URL:|
Kelly-Worden M, Hammer L, Gebhard R, Schrader L, Griffin M, Cooper D. Anti-nuclear antibodies positive serum from systemic lupus erythematosus patients promotes cardiovascular manifestations and the presence of human antibody in the brain. J Pharm Bioall Sci [serial online] 2014 [cited 2020 Aug 10];6:198-204. Available from: http://www.jpbsonline.org/text.asp?2014/6/3/198/135247
Patients with systemic lupus erythematosus (SLE) produce abnormal antibodies that incorrectly identify tissues within the human body instead of a component of an invading organism or virus. A common diagnostic tool in diagnosing lupus is to test for the presence of autoimmune antibodies that bind elements within the nucleus of cells known as antinuclear antibodies (ANAs). ,, ANAs are present in the majority (78-95%) of patients with SLE and there is some correlation between antibodies present and organs affected.
SLE becomes more serious when organs such as the heart, kidneys and/or central nervous system (CNS) are affected. The heart is affected in over 50% of SLE patients. Though all layers of the heart may be affected including the myocardium, pericarditis and heart valve lesions are more prominent. Immune complexes are believed to play a role in the development of the pericarditis and heart valve lesions may be the result of immune deposits left on the valves. Libman-Sacks endocarditis is the most common cardiac manifestation in patients with SLE and primary antiphospholipid syndrome. It is characterized by vegetations associated with the valves, valve thickening or fibrosis, valve regurgitation and rarely valve stenosis. , It is most commonly clinically associated with cardioembolism (predominantly to the brain) and it is difficult to diagnose. 
Murine and rat models exist for the examination of SLE. Rat models include the Lewis rat that has long been used as a model to study autoimmune disorders such as rheumatic fever  experimental autoimmune anterior uveitis,  experimental allergic encephalomyelitis (EAE),  and immune complex disease/serum sickness.  The model for serum sickness in the Lewis rat requires injection with large amounts of bovine serum albumin (BSA). This model generates the formation of immune complexes in the kidney and deposition in the nerve roots with little damage to the nervous system. Replacing BSA with human serum albumin in rats results in the formation of in situ immune complexes in the choroid plexus of the brain and kidneys. 
In this study, we replaced human serum albumin with a human ANA positive serum control obtained from MBL International Corporation and examined Lewis rats for the presence of antibody in the brain as well as the presence of deposits on heart valves.
| Materials and Methods|| |
Animals and experimental groups
Female Lewis rats between 4 and 6 months of age were obtained. Rats were separated into experimental (n = 18 total) and control groups (n = 13 total) and randomly numbered. Animal groups were as follows: (1) ANA positive serum injected (equivalent of ~55 units, MBL international, n = 10 experimental); (2) ANA positive serum and histamine injected (4 mg/kg IP, n = 8 experimental); (3) saline injected (n = 8 controls) and (4) ANA negative control serum injected (MBL international, n = 5). Saline control group rats were injected with saline for a period of either 1 (n = 3) or 3 months (n = 5). At the end of 1 or 3 months, rats were euthanized using carbon dioxide or injected with Inactin 100 mg/kg IP and/or exposed to halothane in mineral oil in a fume hood using a nose cone; a hind limb pull test was performed to ensure the animal was sufficiently anesthetized. Following euthanasia, rat brains, hearts and kidneys were removed and examined for macroscopic lesions. The study design, methods, procedures and protocols were approved per the regulations by Institutional Animal Care Committee and Institutional Review Board at Ball State University, Indiana, USA.
ANA positive and negative serum
ANA positive control serum and ANA negative control serum were each spin filtered and resuspended into a comparatively equal volume of saline before use. This ANA serum is used as a positive standard for ANA testing and displays a homogenous pattern for human ANA antibodies. A negative control group was injected with ANA negative control serum. This serum is a standard negative control serum for ANA testing (MBL International). Injections were ~55 units per animal once a week for a period of 1 month through the tail vein.
Hearts were removed and rinsed in phosphate buffered saline (PBS) and placed in a sucrose storage solution  before storing in a −80°C freezer until use. Hearts were thawed, perfused with PBS and excess fluid was removed before being weighed. Pictures of hearts were taken using a Sony DSC-T200 Cybershot camera with 8.1 Megapixels at 5 × zoom. Each heart was then dissected and separated into atria and ventricular sections. A picture was taken to observe the ventricular wall thickness. The valves were then examined for signs of verrucae with a 5 × 0.25 NA CP-Achromat objective using a microscope with side lighting.
Preparation of samples
Tissue samples frozen in optimal cutting temperature medium were sliced at 20 microns and placed on poly-d-lysine prepared slides. Slides were stored at −22°C until use. Slides were thawed and treated with 4% paraformaldehyde on a rocker for 1 h. Each slide was then washed with PBS (Fisher Scientific) and rocked for 5 min.
Staining with Oil Red O
A 0.5% Oil Red O (CI 26125) stock solution in isopropanol was prepared from working solutions of 60% stock stain in distilled water. Fresh working solutions were prepared each time. Stain was applied for 15 min. Slides were rinsed with 60% isopropanol following staining. Each tissue sample was set with 1,4-diazabicyclo[2.2.2]octane and covered with a cover slip prior to imaging.
Stereotaxically comparative brain slices were acquired from three control and three experimental animals and separated into stereo-equivalent sections (rats 1-3). Brain slices were sorted into (1) hippocampus and amygdala containing regions, (2) hypothalamus containing regions and (3) regions of the corpus callosum and anterior commissure. Slices were treated with anti-human Alexa Fluor 488, for approximately 1 h before washing 3 times in PBS and examining for fluorescence (excitation of 495 nm, emission wavelength of 519 nm) using a microplate reader.
Five rat brains (rats 4-8) were examined for the presence of lesions both externally and after slicing at 90 micrometers. One brain was further examined for the presence of anti-human antibodies using Alexa Fluor 488 and a fluorescent microscope. Slices were treated with anti-human Alexa Fluor 488, for approximately 1 h before washing 3 times in PBS. Slices were further treated with Sudan Black prior to imaging.
Approximately 1 ml of blood was drawn from the tail vein prior to injection to obtain a zero time point. Samples were then taken every 2 weeks after injection and prior to euthanasia. All blood samples were spun for 5 min in a centrifuge at 1700 rpm and 4°C. Plasma layer and blood layer were separated and stored in a −80°C freezer for later use.
Blood plasma samples from three control and three ANA injected rats were examined for histamine levels. Histamine for Rat Enzyme-Linked Immuno-Sorbent Assay (ELISAs) was obtained from Rocky Mountain Diagnostics. ELISAs were performed according to the insert sheet protocol.
| Results|| |
Examination of heart tissue
In order to determine if the heart was affected by ANA injection, hearts were examined for signs of endocarditis (including an increase in deposition) and enlargement. All control hearts (saline injected) were negative for endocarditis. The hearts appeared healthy and not enlarged [Figure 1]a and the valves were normal [Figure 1]b and c. However, ANA injected animals presented with hearts that appeared enlarged with fat deposits around the atria [Figure 2]a which was even greater than that observed associated with negative control hearts [Figure 2]b. Oil Red O staining was performed to examine for further lipid deposits in the hearts. Total red counts were measured in order to quantitate the image data using Image Pro software is by Media Cybernetics, Rockville, MD. USA.
|Figure 1: Control hearts appeared healthy and not enlarged (a) and the valves were normal (b, at white arrow) and (c)|
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|Figure 2: A large amount of fat surrounded the atria of the anti-nuclear antibodies (ANA) positive hearts (a) in comparison to the control hearts [Figure 1] and the negative control hearts (b). Control hearts displayed low levels or red staining (c). However, ANA injected animal hearts presented with red stain even within the myocardium (d)|
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The control hearts [Figure 2]c demonstrated low levels of red in comparison to the ANA positive hearts [Figure 2]d. When statistically examined, ANA injected animal hearts contained significantly greater amounts of red stain than either control hearts or negative control hearts [Table 1].
In order to determine if there was preferential lipid deposits associated with a specific region of the heart, the hearts were separated into three regions (atria [valve], ventricle and apex). We found that there was a significant difference (P < 0.05) in Oil Red O staining between groups. The red intensity was highest for all ANA hearts in the atria and lowest in the apex samples; consistent with normal deposition of fat around the heart [Table 2]. The negative control group had a slightly higher intensity of red staining than the control hearts in the atrial region (105 and 83 respectively). However, it was lower than the intensity found in this region of the ANA positive hearts. Single factor ANOVAs were performed to determine if there was a significant difference between saline control, ANA negative injected control and ANA injected positive hearts. ANA injected hearts were significantly different from both saline injected and ANA negative control injected hearts [Table 3].
When the myocardium was examined, all seven of the ANA positive hearts appeared to display myocardial involvement due to increased ventricular thickening of the ANA injected positive hearts in relation to the control hearts. Reverse examination of three control heart and four ANA heart digital images confirmed that the enlarged hearts appeared significantly larger (ventricular thickness for control heart mean ± standard deviation (SD) =2.20 ± 0.27 and for ANA injected heart, mean ± SD = 3.20 ± 0.25, P = 0.03).
Examination of the valves presented an interesting finding, one of the seven ANA positive hearts examined also presented with the verrucae clusters characteristic of Libman-Sacks endocarditis. The clusters of verrucae were found along the edge of the valve [Figure 3]. The valves of the ANA negative control group were negative for Libman-Sacks endocarditis though, as were the valves of control hearts.
|Figure 3: Valve cusps of an anti-nuclear antibodies positive serum injected rat displaying the presence of verrucae (arrow)|
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Examination of Lewis rat brains
Sections with prominent hippocampus and hypothalamic regions were examined due to damage and/or binding observed within these regions and related regions in marine models and/or in human patients with CNS-SLE or neuropsychiatric-SLE. ,,, Three rat brains from each group were removed and assayed for total human antibody counts using immunofluorescence. ANA injected rats were significantly positive for the presence of human antibody in all sections when compared with controls [Table 4] and over a third (roughly 38%) of ANA injected rats examined developed macroscopic white cerebral lesions that were macroscopically visible on the surface of the rat visual center (occipital lobe equivalent).
|Table 4: Presence of human antibody within the brain of ANA treated rats|
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Control rats, which were injected for 1 month, were free of macroscopic surface brain lesions. Extending injections from 1 to 3 months had no effect on lesion development. Examination of heart and kidneys demonstrated only one minor adrenal anomaly and one report of a right atrial defect. However, rats within the ANA injected group were positive for lesions to the brain (~43%), heart (~14%) and kidneys (~29%).
Both experimental and control groups were examined for the presence of necrotic internal brain lesions as well. Initial inspection demonstrated necrotic lesions in the ANA injected group that were prominently unilateral and were mainly in the region of the caudate/putamen but also included the thalamus, corpus callosum, as well as the hippocampus and superior cortical regions. Only one ANA injected rat brain was free of necrotic lesions. When this brain was sectioned and slices containing caudate/putamen and cerebral cortex were incubated with anti-human secondary (Alexa Fluor 488), the brain fluoresced positive for the presence of anti-human antibody [Figure 4].
|Figure 4: Anti-nuclear antibodies (ANA) injected rat brains were positive for human IgG. Stereotaxically equivalent rat brain slices were treated with Alexa Fluor 488, washed and incubated with Sudan black. (a) Control rat brain slice showing the cudate/putamen (CPu). (b) Control rat brain slice of the cortex. The brain is negative though you can see the mild fluorescence of blood vessels within the brain (line and two fluorescent spots in B) produced by Alexa 488. (c) ANA injected rat brain 4 was positive for human antibodies (green fluorescent speckles) in the caudate/putamen and (d) cortex though the brain appeared normal|
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Serum/plasma samples were also obtained from these rats. Samples were up to ~300 μL in volume. Rats were analyzed for the presence of IgG (n = 3) and histamine (rats n = 3). Circulating ANA values were marginal prior to euthanasia and only slightly greater than controls (t-test of sets, mean ± SD = 1.74 ± 0.05 for control, mean ± SD = 2.03 ± 0.14 for ANA injected P < 0.03 one, P = 0.057 two). It takes only a couple days for rats to remove antibody from the blood stream. Though approximately 55 units were injected each week, only ~2.1 units were detectable in the blood serum by the end of 4 weeks. Therefore, the rat removes the antibodies from its bloodstream with less than a half a unit (0.7%) actually in circulation by the end of the experiment (since control IgG values are ~1.7 units).
However, histamine levels were not significantly different between the human ANA injected group and the saline injected control group (P > 0.05). In fact, histamine levels decreased in the ANA injected group and exhibited less variability than controls. We examined the effects of injecting the animals with histamine prior to ANA injection; these animals did not differ significantly from the experimental ANA injected group for the presence of antibody within the brain. However, the highest level of fluorescence was observed in a slice from an animal of the histamine injected group and this group presented the only case of CNS damage related behavior (hind leg dragging).
| Discussion|| |
The results demonstrate both cardiovascular and neurological pathology in the ANA injected rats. The Lewis rat responds to a foreign protein (antibody) with an increase in circulating immune complexes.  These complexes are actually formed due to the rats own immune response against the human serum antigen. The IgG antibody in the human serum, as well as the rat-anti-human IgG complex, has negative effects on the heart including the valves, leaving small deposits. The buildup of these complexes along the valves can be detrimental due to the inadequate blood supply to the valves of the heart. However, the damage due to IgG alone was not as significant as the damage done by the ANA positive serum. This is consistent with the findings of Kasp et al., where circulating immune complex levels rose earlier with an autoreactive antigen than those against a nonreactive antigen. Due to the increase in autoreactive complexes, there can also be an increased response by the body; which creates more buildup.
ANA injected positive rats also displayed the presence of human IgG in the brain as determined by mean fluorescent values of antibody that were higher than controls and 80% of these animals displayed necrotic lesions within the brain. Though macroscopic external white lesions were predominantly in the visual center, internal lesions were diffuse, predominantly unilateral and mainly in the caudate/putamen region but also affected regions of the thalamus, corpus callosum as well as hippocampal and superior cortical regions.
This is consistent with CNS manifestations observed in SLE patients. Abnormal MRIs are observed in the majority (71%) of patients demonstrating cerebral atrophy and lesions. Lesions are diffuse and hypothesized to be the result of acute changes in the blood brain barrier permeability secondary to vasculopathy, the subsequent result of patchy ischemia and/or the result of anti-neuronal antibodies and cytokines. , Anti-neuronal antibodies have been shown to be associated with active lupus encephalopathy; and the presence of these antibodies is consistent with the diffuse nature of CNS lesions.  Lesions within neuropsychiatric-SLE patients are found in both grey and white matter of the cerebellum, cerebrum or brainstem.  However, significant damage has been observed in areas within the limbic system such as the amygdala and hippocampus as well. ,
Animals receiving ANA serum alone did not display signs of EAE such as hind limb dragging. However, one of the rats receiving ANA positive serum in conjunction with histamine injection did display this behavior. Though there was no significant difference in fluorescent levels for the presence of human antibody in the brain between ANA serum alone and ANA serum with histamine injection, the histamine injected group possessed the highest level of fluorescence. This was to be expected since histamine is known to increase blood brain barrier permeability through H2 receptor stimulation. 
Plasma histamine levels of the control rats displayed a wide variety of values and this may explain why there was no difference between ANA serum injected and controls. However, there was an unusual decrease in histamine values for rats that received ANA serum injections. It is possible that cytokines in the serum promote a down-regulation of the allergic response in the promotion of the inflammatory response. Interferons are a family of antiviral proteins that promote differentiation of monocytes into dendritic cells that capture antigen and present them to CD4 + T cells, promote NK cell activity against tumors and regulate B-cell proliferation and production of IgG as well as other functions. , Patients with SLE present with interferon-alpha (IFN-alpha) in their plasma. IFN-alpha/beta enhances IgE dependent secretion of histamine from human peripheral blood basophils. However, INF-alpha/beta inhibits IgE-dependent histamine release from mast cells in the rat.  Therefore, it is possible that histamine release actually decreases due to the presence of IFN-alpha in the ANA positive serum from SLE patients. This would also explain why the only rat which displayed an EAE response was in the group receiving histamine injections. However, this could also be attributed to histamine depletion that occurred after the initial burst of histamine release from mast cells including those present in the CNS. Depletion of histamine stores has been proposed in patients with multiple sclerosis (MS) and histamine injection has been used to treat patients with MS. Treatment has improved some symptoms. However, as noted earlier, blood brain barrier permeability would also be increased with histamine administration.  Therefore, a burst of histamine release would deplete histamine stores while increasing blood brain barrier permeability and infiltration of antibody into the brain.
Finally, together the heart and brain are affected by the presence of the positive ANA human serum. The affect on the heart is consistent with what is known of the effects of SLE on the heart. Libman-Sacks endocarditis, the most common cardiac manifestation in patients with SLE and primary antiphospholipid syndrome, was also observed. , Libman-Sacks endocarditis is also commonly clinically associated with cardioembolism to the brain.  The association of cardioembolism to the brain could promote vascular damage to the brain, increased vascular permeability and movement of antibody into the brain. Thus, cardiac involvement in SLE could be a precursor to CNS manifestations. Future research may address this hypothesis.
The results of this study must be viewed in light of potential limitations. First, we had a small sample size. This could limit the statistical inferences and validity of the findings. However, it should be noted that a pilot study was conducted first to determine that significance could be achieved with this sample size.
Second, as characteristic of any experiment, the control and experimental groups may have individual inherent differences in physiologic function. If so, this may have led to a bias in findings. However, the control and experimental rat groups are the same species of rat and where obtained from a single colony source.
Finally, as with any type of research, even though the utmost care was taken to conduct the experiment, there may have been inadvertent experimental errors.
| Conclusion|| |
As is consistent with serum sickness and SLE, damage to multiple organs was observed. The animals displayed damage to not only the brain, but to the heart and kidneys as well. The presence of lesions in the heart is consistent with damage to the heart and it is of special interest that Libman-Sack's endocarditis was observed in one of the ANA serum injected animals. Libman-Sacks are a nonbacterial carditis that results from immune complex formation. Though the prevalence of Libman-Sacks endocarditis in SLE is unknown due to the difficulty in diagnosing the disorder, Libman-Sacks endocarditis has been observed during numerous autopsies and postmortem studies of patients with SLE. ,, Though further investigation will need to be done in order to determine cell infiltrates into damaged regions, we can conclude that human ANA antibodies do cross over the blood brain barrier in the Lewis rat and that both heart and brain manifestations are present consistent with what is observed in SLE.
| Acknowledgments|| |
We would like to acknowledge Heather Bruns, Najma Javed, Scott Pattison, Courtney Huff, and Sara Ghassemifar for individual contributions to this project. This project was funded through Ball State University ASPiRE grants, the Ball State Chapter of Sigma Xi and our laboratory's Neuroscience Research account.
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[Figure 1], [Figure 2], [Figure 3], [Figure 4]
[Table 1], [Table 2], [Table 3], [Table 4]
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