Degree of modification of Ro60 by the lipid peroxidation by-product 4-hydroxy-2-nonenal may differentially induce Sjögren syndrome or systemic lupus erythematosus in BALB/c mice

Abstract

Our previous work showed that immunization of rabbits with 4-hydroxy-2-nonenal-modified Ro60 (HNE-Ro60) accelerates autoimmunity. We extended this model into mice, hypothesizing that the severity of autoimmunity would be dependent on the degree of HNE modification of Ro60. Five groups of BALB/c mice (10/group) were used. Group I was immunized with Ro60. Groups II to IV were immunized with Ro60 modified with 0.4 mM (low), 2 mM (medium), and 10 mM (high) HNE, respectively. Group V controls received Freund's adjuvant. A rapid abrogation of tolerance to Ro60/La antigens occurred in mice immunized with HNE-modified Ro60, especially in the low and medium HNE-Ro60 groups. Lymphocytic infiltration and significantly high decrement in salivary flow (37%) compared to controls was observed only in the high HNE-Ro60 group, suggesting induction of a Sjögren syndrome-like condition in this group. Anti-dsDNA occurred only in mice immunized with medium HNE-Ro60. This group did not have a significant decrement in salivary flow, suggesting induction of a systemic lupus erythematosus-like manifestation in this group. Significantly high antibodies to Ro60 were found in saliva of mice in the low and medium HNE-Ro60 and the Ro60 groups, as well as anti-HNE Ro60 in the low and medium HNE-Ro60 groups. Understanding the mechanism of this differential induction may help discriminate between these two autoimmune diseases.

Figure 1

ELISA results of Ro60, used as solid phase antigen, bound by mice bleeds 1-4. Group I-mice immunized with unmodified Ro60; Group II-mice immunized with low HNE Ro60; Group III-mice immunized with moderate HNE Ro60; Group IV-mice immunized with high HNE Ro60; Group V-mice immunized with Freund's alone. CC refers to conjugate control. P refers to positive control and N refers to normal control.

Figure 2

ELISA results of low HNE Ro60, used as solid phase antigen, bound by mice bleeds 1-4. Group I-mice immunized with unmodified Ro60; Group II-mice immunized with low HNE Ro60; Group III-mice immunized with moderate HNE Ro60; Group IV-mice immunized with high HNE Ro60; Group V-mice immunized with Freund's alone. CC refers to conjugate control. P refers to positive control and N refers to normal control.

Figure 3

ELISA results of moderate HNE Ro60, used as solid phase antigen, bound by mice bleeds 1-4. Group I-mice immunized with unmodified Ro60; Group II-mice immunized with low HNE Ro60; Group III-mice immunized with moderate HNE Ro60; Group IV-mice immunized with high HNE Ro60; Group V-mice immunized with Freund's alone. CC refers to conjugate control. P refers to positive control and N refers to normal control.

Figure 4

ELISA results of high HNE Ro60, used as solid phase antigen, bound by mice bleeds 1-4. Group I-mice immunized with unmodified Ro60; Group II-mice immunized with low HNE Ro60; Group III-mice immunized with moderate HNE Ro60; Group IV-mice immunized with high HNE Ro60; Group V-mice immunized with Freund's alone. CC refers to conjugate control. P refers to positive control and N refers to normal control.

Figure 5

Immunoblot analysis of intermolecular epitope spreading to La autoantigen in the Ro60, Freund's adjuvant and low, medium or high HNE-modified Ro60 immunized animals. Immunoblotting was carried as mentioned in “Materials and methods”. Top panel-Bleed 1; Bottom panel-Bleed 2.

Figure 6

ELISA analysis of intermolecular epitope spreading to La autoantigen in the Ro60, Freund's adjuvant and low, medium or high HNE-modified Ro60 immunized animals. Top panel-Bleed 1; Bottom panel-Bleed 2. ELISA was carried out as mentioned in “Materials and methods”. Values are means ± standard deviation (8 animals in Ro 60 group and Freund's control; 10 animals each in the other three groups), a= p<0.000046 compared to high HNE Ro60 group, b= p< 0.00021 compared to Freund's control group, c= p< 0.053 compared to Ro60 group, d=p< 0.056 compared to low HNE group, e= p< 0.00006 compared to high HNE group, f= p< 0.000158 compared to Freund's control group

Figure 7

Antibodies to dsDNA detected by CLIF assay in mice immunized with Ro60, Freund's adjuvant and low, medium or high HNE-modified Ro60. “+” refers to human positive control, and “-” to human negative control supplied in the kit. A: Ro60 immunized group; B: Low HNE Ro60 group. C: moderate-HNE Ro60 group; D: high-HNE Ro60 group. E: Freund's control

Figure 8

Salivary flow in mice immunized with unmodified Ro60, low HNE Ro60, medium HNE Ro 60, high HNE Ro60 and Freund's adjuvant only. Minimum, 25%tile, median, 75%tile and maximum are shown with 10 animals in each group. Salivary flow is measured as mentioned in “Materials and methods”., a= p<0.05 compared to Ro60 group, Kruskal-Wallis rank order testing, b= p< 0.05 compared to Freund's control group, Kruskal-Wallis rank order testing

Figure 9

Pathology of salivary glands from animals immunized with high HNE Ro60 or Freund's only. Salivary lymphocytic infiltrates in an animal immunized with high HNE-modified Ro60 (Top Panel) and in an animal immunized with Freund's (Bottom panel).

Figure 10

ELISA analysis of anti Ro60 antibodies in the saliva of mice immunized with Ro60, Freund's adjuvant and low, medium or high HNE-modified Ro60 (Groups I-V) using unmodified Ro60 as solid phase substrate. Group I: Mice immunized with unmodified Ro60; Group II: Mice immunized with Ro60 modified with 0.2 mM HNE (low HNE); Group III: Mice immunized Ro60 modified with 2 mM HNE (medium HNE); Group IV: Mice immunized Ro60 modified with 10 mM HNE (high HNE); Group V: Mice immunized with Freund's alone. Antibody response is evident in Ro, HNE 1 and 2 groups. Values are means ± SD for 4 determinations.

Figure 11

ELISA analysis of anti-HNE Ro60 antibodies in the saliva of mice immunized with Ro60, Freund's adjuvant and low, medium or high HNE-modified Ro60 (Groups I-V) using high HNE modified Ro60 as solid phase substrate. Group I: Mice immunized with unmodified Ro60; Group II: Mice immunized with Ro60 modified with 0.2 mM HNE (low HNE); Group III: Mice immunized Ro60 modified with 2 mM HNE (medium HNE); Group IV: Mice immunized Ro60 modified with 10 mM HNE (high HNE); Group V: Mice immunized with Freund's alone. Antibody response is evident in Ro, HNE 1 and 2 groups. Values are means ± SD for 4 determinations.

Figure 12

Anti-HNE levels in the first bleed of mice immunized with unmodified Ro60, low HNE Ro60, medium HNE Ro60, high HNE Ro60 and Freund's adjuvant. Values are means ± standard deviation (10 animals in Ro60 and Freund's control group; 3 animals in HNE Ro60 groups). p<0.004 –anti-HNE levels in medium HNE Ro60 group versus low HNE Ro60 group

Figure 13

Anti-HNE levels in the first and seconds bleeds of mice immunized with unmodified Ro60, low HNE Ro60, medium HNE Ro60, high HNE Ro60 and Freund's adjuvant. Values are means ± standard deviation (8 animals in Ro 60 and Freund's control group; 10 animals in HNE Ro60 groups).