Antibody induction of lupus-like neuropsychiatric manifestations

Abstract

Although systemic lupus erythematosus (SLE) is usually evaluated with regard to autoimmune reactivity toward the kidney, there are multiple psychiatric abnormalities associated with this autoimmune disease. Lupus-prone male NZM88 mice, derived from NZB/NZW F1 mice, develop early neuropsychiatric manifestations without any signs of nephritis. In addition to the usual repertoire of antibody specificities, including autoantibodies to dsDNA and renal antigens, mice of this inbred strain express autoantibodies to numerous brain antigens. Here, we show that autoantibodies to brain antigens, assessed by Western analysis, are as individually varied as are the diverse neuropsychiatric manifestations observed in SLE patients. Additionally, a monoclonal antibody derived from the spleen of an untreated NZM88 male when injected into healthy BALB/cByJ, but not C57BL/6J, mice induced behaviors similar to those of lupus-prone NZM88 mice. This monoclonal antibody, which is specific to dynamin-1, binds preferentially in BALB/cByJ cortex and induces substantial expression of cytokines mainly in the hypothalamus. Thus, an antibody to just one brain antigen can induce multiple behavioral changes, and multiple autoantibodies to different brain antigens exist in lupus-prone mice; however, susceptibility to the induction of neurobehavioral deficits is dependent on host genetics.

Figure 1

Behavior of C57BL/6 (B6) mice and four NZM strains in Morris water maze during training (A) and probe trial (B). Six males per NZM strain and 17 B6 males were each assayed in eight sessions over a 4 day period. The time required to find the platform in each session is shown (A). One hr after the final training trial, the platform was removed, and the probe trial was performed for 60 sec (B). Data is reported as mean and standard error of mean. In Morris water maze sessions (A), NZM88 mice learning curve was significantly different from that of the B6, 391 and 2328 strains. In the probe trial (B), each bar represents the mean ± standard error of mean for the amount of time (sec) spent in the actual area of the platform prior to it's removal within the 60 sec trial; bars labeled with a different letter are significantly (p < 0.05) different. All NZM strains were statistically different from the B6 strain, and the NZM88 strain was statistically different from the other NZM strains.

Figure 2

Exploratory activity of male NZM mice. Shown are time spent in the inner circle of the open-field chamber (A); number of squares crossed (B), and number of wall rears (C), during a 5 min observation period. Each bar represents the mean ± standard error of mean for each NZM strain performing the specified activity; bars labeled with a different letter are significantly (p < 0.05) different.

Figure 3

Brain-reactive antibodies. Western analysis of antibodies to brain antigens (brain from BALB/cByJ SCID mouse) with sera from a female NZM88 mouse (lane 3), male NZM88 mice (lanes 4-16), and female and male BALB/cByJ mice (lanes 1, 2, 17, and 18).

Figure 4

Assessment of SB31 specificity for brain antigens. Immunoprecipitation of brain antigens with a brain homogenate from a BALB/cByJ SCID mouse by SB31 plus protein G-agarose followed by SDS-PAGE and SYPRO Ruby staining (a); the arrow indicates the band extracted for MS/MS analysis. In b, Western analysis of a BALB/cByJ mouse brain homogenate was performed with SB31 as the primary antibody followed by HRP conjugated goat-mIgG.

Figure 5

SB31 recognizes the brain protein dynamin-1. A BALB/cByJ mouse brain homogenate was immunoprecipitated with NB28, a monoclonal anti-dynamin-1 (OncogeneResearch Products, Boston, MA), and the blotted proteins were probed with SB31 followed by biotinylated goat anti-mouse IgG and streptavidin-HRP. The arrow indicates dynamin-1.

Figure 6

Behavioral changes induced by SB31 anti-dynamin-1. Healthy male and female BALB/cByJ mice were injected once-weekly intravenously with IgG1 control or SB31 (100 μg/injection). After 5 wk, mice were assayed. Activity in a dark open field was monitored for 15 min and the following were measured: data shown are total distance (cm) traveled (a), percentage of time spent in the center (b), and percentage of distance traveled in center (c). Behavior in the elevated plus maze was measured, as percentage of time spent in open arms (d) and percentage of entries into the open arms (e).

Figure 7

Brain regional binding of SB31. One wk after the third weekly intravenous injection of SB31 or isotype control IgG2a (PC5) into BALB/c or B6 male SCID mice, the brain regions were isolated from saline-perfused mice and assayed by ELISA. Each bar represents the mean of two mice. The results were relatively similar with a repeated experiment.

Figure 8

Immunohistochemistry of SB31 (anti-dynamin-1) binding to coronal sections of cerebellum and cortex of a BALB/cByJ brain after the fifth weekly intravenous injection of SB31. Strong cerebellar SB31 immunofluorescence (A) is seen in the Purkinje cell layer (PCL), but not the molecular (MCL) or granule (GCL) cell layers. The level of binding of SB31 in the cerebellum was near equivalent after the third weekly injection. Intense staining may reflect synaptic contacts outlining the Purkinje cell bodies. Control binding assessed with an irrelevant IgG2a (PC5) displayed no selective binding (B, cerebellum; D, cortex). Binding of SB31 in cortex shows discrete binding throughout the cortex (C).

Figure 9

Light/Dark Box behavioral test for anxiety of NZM strains and BALB/c mice treated with anti-dynamin-1. Latency in crossing into the light half of a light/dark box for NZM males (A) or anti-dynamin-1 (SB31) or isotype control (PC5)-injected BALB/c males (B), and time in light half of the box during a 5 min evaluation period for NZM males (C) and injected BALB/c males (D). Each bar represents the mean ± standard error of mean for each NZM strain performing the specified activity; bars labeled with a different letter are significantly (p < 0.05) different; BALB/c differences are noted.