β2-microglobulin is required for the full expression of xenobiotic-induced systemic autoimmunity

Figures & data

Table 1.  Immunoglobulin levels and autoantibodies in B2m^+/+, B2m^+/−, and B2m^−/− mice following 4 weeks of mercury exposure^a.

B2m	No.	Treatment	Immunoglobulin level		ANoA	Anti-chromatin
			IgG1	IgG2a
			(mg/mL)	(mg/mL)	(Pos/No)	OD405
+/+	8	PBS	0.250 ± 0.060	0.113 ± 0.025	0/8	0.05 ± 0.02
	9	HgCl2	1.106 ± 0.292	0.208 ± 0.080	9/9	0.73 ± 0.46
+/-	10	PBS	0.269 ± 0.092	0.091 ± 0.034	0/10	0.07 ± 0.09
	8	HgCl2	1.079 ± 0.256	0.182 ± 0.078	6/8	0.46 ± 0.29
−/−	8	PBS	0.130 ± 0.107	0.015 ± 0.003	0/8	0.04 ± 0.06
	8	HgCl2	0.174 ± 0.165	0.021 ± 0.007	8/8	0.75 ± 0.57

^aValues are expressed as mean (± 1 SD); p values were determined for comparisons between PBS and HgCl₂ data within each group, and between HgCl₂ data between groups; NS, not significant.

IgG₁, PBS vs. HgCl₂ B2m^+/+, p < 0.0001; B2m^+/−, p < 0.0001; B2m^−/−, NS.

IgG_2a, PBS vs. HgCl₂ B2m^+/+, p < 0.006; B2m^+/−, p < 0.004; B2m^−/−, NS.

Anti-chromatin Ab, PBS vs. HgCl₂ B2m^+/+, p < 0.001; B2m^+/− p < 0.001; B2m^−/−, p < 0.004.

IgG₁, HgCl₂ B2m^+/+ vs. B2m^−/−, p < 0.0001; B2m^+/− vs. B2m^−/−, p < 0.0001.

IgG_2a, HgCl₂ B2m^+/+ vs. B2m^−/−, p < 0.0001; B2m^+/− vs. B2m^−/−, p < 0.0001.

Anti-chromatin Ab, HgCl₂ B2m^+/+ vs. B2m^−/−, NS; B2m^+/− vs. B2m^−/−, NS.

Table 2.  Immunopathological changes in B2m^+/+, B2m^+/−, and B2m^−/− mice following 4 weeks of mercury exposure^a.

B2m	No.	Treatment	Kidney immunopathology				Spleen immunopathology
			Glomerular IgG	Glomerular C3	Vessel IgG	Vessel C3	Vessel IgG	Vessel C3
+/+	8	PBS	0	30 ± 60	0	0	0	0
+/+	9	HgCl2	978 ± 726	676 ± 485	0.45 ± 0.88	0.11 ± 0.33	1.33 ± 0.87	0.89 ± 0.55
+/−	10	PBS	90 ± 224	120 ± 222	0.06 ± 0.18	0	0.06 ± 0.18	0.06 ± 0.18
+/−	8	HgCl2	710 ± 512	790 ± 548	0	0	0.25 ± 0.46	0.19 ± 0.26
−/−	8	PBS	0	0	0	0	0	0
−/−	8	HgCl2	760 ± 559	560 ± 468	0.25 ± 0.71	0.06 ± 0.18	0.44 ± 1.05	0.25 ± 0.71

^aValues are expressed as mean (± 1 SD); p values were determined for comparisons between post-PBS and post-HgCl₂ data within each group, and between post-HgCl₂ data between groups.

Glomerular IgG, post-PBS vs. post-HgCl₂ B2m^+/+, p < 0.001; B2m^+/−, p < 0.001; B2m^−/−, p < 0.001.

Glomerular C3, post-PBS vs. post-HgCl₂ H- B2m^+/+, p < 0.001; B2m^+/−, p < 0.005; B2m^−/−, p < 0.001.

Kidney vessel IgG, post-PBS vs. post-HgCl₂ B2m^+/+, NS; B2m^+/−, NS; B2m^−/−, NS.

Kidney vessel C3, post-PBS vs. post-HgCl₂ B2m^+/+, NS; B2m^+/−, NS; B2m^−/−, NS.

Splenic vessel IgG, post-PBS vs. post-HgCl₂ B2m^+/+, p < 0.005; B2m^+/−, NS; B2m^−/−, NS.

Splenic vessel C3, post-PBS vs. post-HgCl₂ B2m^+/+, p < 0.005; B2m^+/−, NS; B2m^−/−, NS.

Splenic vessel IgG, post-HgCl₂ B2m^+/+ vs. B2m^−/−, p = 0.0266; post-HgCl₂ B2m^+/+ vs. B2m^+/−, p = 0.0133.

Splenic vessel C3, post-HgCl₂ B2m^+/+ vs. B2m^−/−, p = 0.0234; post-HgCl₂ B2m^+/+ vs. B2m^+/−, p = 0.0089.

Figure 1.  ANA and ANoA in HgCl₂-exposed B2m^+/+ and B2m^−/− mice. Mice were injected with HgCl₂ (40 µg in 100 µL PBS) twice per week for 4 weeks before determination of serum IgG ANoA by indirect immunofluorescence on HEp-2 cells. (A) Endpoint titer of IgG ANoA in B2m^+/+ and B2m^−/− mice. The geometric mean (and range) of IgG ANoA titers differed significantly between B2m^−/− and B2m^+/+ mice (340 [160–640] vs. 1540 [160–2560]; p < 0.025). (B) Fluorescence intensity of IgG ANA and ANoA in B2m^+/+ (n = 9), B2m^+/− (n = 8) and B2m^−/− (n = 8) mice at a serum dilution of 1/100. ANoA autoantibodies (as mean ± SEM) differed significantly between B2m^−/− (n = 8) and B2m^+/+ (n = 9) mice (1.3 (± 1.1) vs. 2.8 (± 0.56); p = 0.0027).

Table 3.  IgG subclass of anti-chromatin antibodies in B2m^+/+and B2m^−/− mice following 4 weeks of mercury exposure.

	IgG subclass of anti-chromatin antibodies (OD405)
	IgG1	IgG2a	IgG2b	IgG3
B2m^+/+	0.63 ± 0.21	1.02 ± 0.64	1.67 ± 1.51	0.03 ± 0.02
B2m^−/−	0.19 ± 0.10	0.60 ± 0.36	1.32 ± 0.98	0.08 ± 0.16
p value	p = 0.0006	p = 0.24	p = 0.96	p = 0.29

Figure 2.  Titration of IgG anti-chromatin antibodies in HgCl₂-exposed B2m^+/+ and B2m^−/− mice. Mice were injected with HgCl₂ (40 µg in 100 µL PBS) twice per week for 4 weeks before determination of serum IgG anti-chromatin antibodies using ELISA. Titration consisted of serial 2-fold dilution of individual sera from 1/100 to 1/3200. OD₄₀₅ values represent the geometric mean at each serum dilution for B2m^+/+ (n = 9) and B2m^−/− (n = 6) mice. B2m^+/+ ▪ B2m^−/− ▪.

Figure 3.  IFNγ, IL-2, and IL-4 expression in the (A) spleen, (B) draining lymph node, and (C) skin of HgCl₂-exposed B2m^+/+ and B2m^−/− mice. Mice were injected subcutaneously with 40 µg HgCl₂ in phosphate-buffered saline (PBS) (solid bars) or with PBS alone (open bars) for 2 weeks. Total RNA was prepared and quantitative real-time polymerase chain reaction was performed on reverse-transcribed RNA. The mRNA level was normalized using cyclophilin A for spleen and lymph node. For skin samples, cytokine mRNA levels were related to total RNA concentration. Values represent the mean and SEM of 4 mice per group.