Effects of lactational exposure to low-dose BaP on allergic and non-allergic immune responses in mice offspring

Figures & data

Figure 1. Experimental design.

Table 1. Number of inflammatory cells in bronchoalveolar lavage (BAL) fluid and pulmonary histopathological findings in male offspring.

	Cell number in BAL fluid (×10^4)				Inflammation score (H&E)	Goblet cell hyperplasia score (PAS)
Group	Macrophages	Neutrophils	Eosinophils	Lymphocytes
Vehicle	17.1 ± 3.31	0.075 ± 0.043	0.003 ± 0.003	0.024 ± 0.009	ND	ND
BaP-L	25.0 ± 3.12	0.038 ± 0.016	0.017 ± 0.017	0.030 ± 0.020	ND	ND
BaP-M	19.8 ± 3.50	0.154 ± 0.067	0.000 ± 0.000	0.009 ± 0.009	ND	ND
BaP-H	17.8 ± 2.63	0.195 ± 0.166	0.000 ± 0.000	0.000 ± 0.000	ND	ND
OVA	25.4 ± 2.99	0.838 ± 0.370**	1.111 ± 0.514**	0.860 ± 0.232**	1.90 ± 0.332	1.30 ± 0.200
OVA + BaP-L	31.3 ± 2.31**	1.899 ± 0.356**	2.648 ± 0.803**	1.429 ± 0.257**	2.70 ± 0.300	1.60 ± 0.245
OVA + BaP-M	22.2 ± 2.48	0.786 ± 0.202**	0.944 ± 0.230**	0.489 ± 0.152**	2.60 ± 0.292	2.20 ± 0.200^#
OVA + BaP-H	24.9 ± 3.74	0.899 ± 0.186**	0.593 ± 0.228**	0.636 ± 0.197**	2.60 ± 0.245	1.60 ± 0.245

Number of inflammatory cells in BAL fluid and histopathological findings in lung were evaluated 24 h after the final OVA instillation. Data shown are means ± SE for 4–8 animals/group for cell number in BAL fluid and for five animals/group for pulmonary histopathological findings, respectively.

** p < 0.01 vs. vehicle group, ^#p < 0.05 vs. OVA group.

ND: no data.

Table 2. Number of inflammatory cells in the bronchoalveolar lavage (BAL) fluid and pulmonary histopathological findings in female offspring.

	Cell number in BAL fluid (×10^4)				Inflammation score (H&E)	Goblet cell hyperplasia score (PAS)
Group	Macrophages	Neutrophils	Eosinophils	Lymphocytes
Vehicle	17.3 ± 1.81	0.136 ± 0.096	0.000 ± 0.000	0.013 ± 0.008	ND	ND
BaP-L	20.9 ± 4.03	0.072 ± 0.035	0.032 ± 0.023	0.019 ± 0.019	ND	ND
BaP-M	20.9 ± 3.23	0.102 ± 0.007	0.000 ± 0.000	0.000 ± 0.000	ND	ND
BaP-H	24.4 ± 4.36	0.172 ± 0.101	0.000 ± 0.000	0.019 ± 0.019	ND	ND
OVA	27.7 ± 3.42**	3.360 ± 0.758**	3.193 ± 1.035**	2.411 ± 0.574**	3.30 ± 0.300	2.25 ± 0.292
OVA + BaP-L	32.6 ± 2.63**	4.072 ± 0.514**	3.694 ± 0.779**	2.966 ± 0.330**	4.30 ± 0.300	3.50 ± 0.224^#
OVA + BaP-M	30.3 ± 1.45**	3.547 ± 0.502**	4.519 ± 0.764**	2.551 ± 0.412**	2.80 ± 0.255	2.00 ± 0.224
OVA + BaP-H	35.2 ± 1.81**	3.418 ± 0.369**	3.587 ± 0.036**	1.728 ± 0.303**	2.86 ± 0.125	2.00 ± 0.289

Number of inflammatory cells in BAL fluid and histopathological findings in lung were evaluated 24 h after the final OVA instillation. Data shown are means ± SE for 4–8 animals/group for cell number in BAL fluid and for five animals/group for pulmonary histopathological findings, respectively.

** p < 0.01 vs. vehicle group, ^#p < 0.05 vs. OVA group.

ND: no data.

Table 3. Protein levels of Th1/Th2 cytokines in the lungs following exposure to BaP on allergic airway inflammation in male offspring.

		pg in total lung supernatants
Group	N	IL-4	IL-5	IL-13	IL-33	IFN-γ
Vehicle	7	91.3 ± 8.56	3.95 ± 0.33	0.35 ± 0.33	5377 ± 180	3.57 ± 0.41
BaP-L	5	98.0 ± 15.3	3.45 ± 0.43	4.25 ± 0.75	5620 ± 565	4.36 ± 0.99
BaP-M	4	65.0 ± 6.08	3.66 ± 1.15	4.33 ± 0.75	5252 ± 239	2.98 ± 0.32
BaP-H	4	68.8 ± 2.87	2.26 ± 0.10	0.96 ± 0.90	5206 ± 124	4.50 ± 0.87
OVA	8	77.0 ± 9.40	175 ± 17.7**	340 ± 49.4**	13225 ± 151**	5.11 ± 0.76
OVA + BaP-L	8	63.8 ± 15.5	319 ± 62.9**	394 ± 71.0**	15902 ± 1659**	5.66 ± 0.44*
OVA + BaP-M	8	63.7 ± 8.58	211 ± 49.4**	263 ± 57.5**	11902 ± 1468**	5.12 ± 0.38
OVA + BaP-H	8	64.0 ± 3.76	190 ± 35.6**	270 ± 51.0**	15354 ± 1422**	4.58 ± 0.42

The supernatants of homogenized lungs were measured by ELISA. Data are shown as means ± SEM of 4 or 8 animals per group.

* p < 0.05.

** p < 0.01 vs. vehicle group.

Table 4. Protein levels of Th1/Th2 cytokines in the lungs following exposure to BaP on allergic airway inflammation in female offspring.

		pg in total lung supernatants
Group	N	IL-4	IL-5	IL-13	IL-33	IFN-γ
Vehicle	7	48.4 ± 6.89	11.0 ± 0.27	0.00 ± 0.00	5369 ± 406	6.48 ± 0.82
BaP-L	5	63.7 ± 14.9	11.0 ± 0.35	0.23 ± 0.23	4720 ± 295	5.80 ± 0.86
BaP-M	5	35.2 ± 7.63	10.4 ± 0.31	0.08 ± 0.08	6492 ± 1530	5.54 ± 0.49
BaP-H	4	57.3 ± 8.12	11.6 ± 0.34	0.00 ± 0.00	4885 ± 335	5.75 ± 0.80
OVA	8	67.7 ± 6.10	372 ± 71.7**	332 ± 56.8**	23218 ± 2249**	8.47 ± 0.63**
OVA + BaP-L	8	56.6 ± 5.71*	553 ± 83.6**	447 ± 55.4**	29794 ± 3143**	10.8 ± 0.65**^,#
OVA + BaP-M	8	49.6 ± 6.60	492 ± 30.6**	396 ± 29.8**	33850 ± 2300**^,#	10.5 ± 0.49**
OVA + BaP-H	8	68.8 ± 7.12	479 ± 36.3**	414 ± 20.0**	29190 ± 2329**	9.52 ± 0.90**

The supernatants of homogenized lungs were measured by ELISA. Data are shown as means ± SEM of 4 or 8 animals per group.

* p < 0.05.

** p < 0.01 vs. Vehicle group.

^# p < 0.05 vs. OVA group.

Figure 2. Ovalbumin (OVA)-specific Ig antibody levels in serum following lactational expo-sure to benzo[a]pyrene in allergic airway inflammation in offspring. Serum OVA-specific IgE and IgG₁ levels were assessed 24 h after the respective final OVA instillation. Data shown are means ± SE for 4–8 animals/group. **p < 0.01 vs. vehicle group.

Figure 3. Total cell number and cell surface molecule expression in mediastinal lymph node cells: male offspring. Data is from FACS sorting of cells harvested 24 h after final intratracheal instillation. (A) Total cell number. (B–D) ovalbumin (OVA)⁻ groups. (E–G) OVA⁺ groups. (B,E) CD11c⁺PDCA-1⁺ and CD11c⁺PDCA-1⁻ cells. (C,F) CD28⁺, TCRβ⁺, and CD28⁺TCRβ⁺ cells. (D,G) CD86⁺, MHC Class II⁺, and MHC Class II⁺CD86⁺ cells. Data shown are means ± SEM of 4–8 animals for total cell number or 4 or 5 animals for FACS analysis, respectively. **p < 0.01 vs. vehicle group, ^#p < 0.05 vs. OVA group.

Figure 4. Total cell number and cell surface molecule expression in mediastinal lymph node cells: female offspring. Data is from FACS sorting of cells harvested 24 h after final intratracheal instillation. (A) Total cell number. (B–D) ovalbumin (OVA)(−) groups. (E–G) OVA(+) groups. (B,E) CD11c⁺PDCA-1⁺ and CD11c⁺PDCA-1⁻ cells. (C,F) CD28⁺, TCRβ⁺, and CD28⁺TCRβ⁺ cells. (D,G) CD86⁺, MHC Class II⁺, and MHC Class II⁺CD86⁺ cells. Data shown are means ± SEM of 4–8 animals for total cell number or 4 animals for FACS analysis, respectively. *p < 0.05, **p < 0.01 vs. vehicle group, ^#p < 0.05 vs. OVA group.

Table 5. Proliferation/Activation of mediastinal lymph node cells in male offspring.

	OVA re-stimulation	Cell proliferation (ABS)	Cytokine expression in culture supernatant (pg/ml)
Group	IL-4	IL-5	IFN-γ
OVA	−	1.03 ± 0.035	5.90 ± 1.48	1.78 ± 0.97	1.19 ± 0.66
OVA + BaP-L		0.90 ± 0.052	5.51 ± 0.66	1.09 ± 0.40	3.81 ± 1.71
OVA + BaP-M		1.01 ± 0.035	9.88 ± 0.98*	2.71 ± 1.28	7.44 ± 1.11*
OVA + BaP-H		0.97 ± 0.068	7.60 ± 1.18	1.12 ± 0.57	4.81 ± 0.21*
OVA	+	1.35 ± 0.089	30.3 ± 7.35	71.8 ± 22.9	14.4 ± 6.78
OVA + BaP-L		1.23 ± 0.108	39.6 ± 18.1	72.5 ± 26.0	11.2 ± 6.09
OVA + BaP-M		1.34 ± 0.072	40.3 ± 3.95	79.4 ± 26.3	8.57 ± 3.57
OVA + BaP-H		1.44 ± 0.129	27.1 ± 7.71	60.6 ± 28.3	6.89 ± 4.56

Cell proliferation and cytokine expression in culture supernatants were examined after 45 h of culture with or without of ovalbumin (OVA) re-stimulation in OVA-sensitized groups. Data expressed in terms of pg/ml culture medium. Data shown are means ± SEM of 4–5 animals/group.

* p < 0.05 vs. OVA group without OVA re-stimulation.

Table 6. Proliferation/Activation of mediastinal lymph node cells in female offspring.

Group	OVA re-stimulation	Cell proliferation (ABS)	Cytokine expression in culture supernatant (pg/ml)
			IL-4	IL-5	IFN-γ
OVA	−	0.86 ± 0.028	2.90 ± 0.70	9.52 ± 2.45	3.71 ± 0.73
OVA + BaP-L		0.87 ± 0.070	8.27 ± 0.75**	13.1 ± 4.75	8.97 ± 1.89
OVA + BaP-M		1.07 ± 0.116	9.00 ± 0.54**	14.4 ± 4.70	8.27 ± 0.64**
OVA + BaP-H		1.10 ± 0.138	9.56 ± 0.73**	6.89 ± 1.36	4.70 ± 0.57*
OVA	+	1.52 ± 0.039	115 ± 23.2	368 ± 69.1	46.5 ± 12.5
OVA + BaP-L		1.64 ± 0.078	141 ± 44.7	370 ± 116	30.8 ± 7.55
OVA + BaP-M		1.76 ± 0.088^#	170 ± 43.8	494 ± 99.1	65.4 ± 13.9
OVA + BaP-H		1.65 ± 0.044	110 ± 12.6	304 ± 20.9	31.6 ± 0.78

Cell proliferation and cytokine expression in culture supernatants were examined after 45 h of culture with or without of ovalbumin (OVA) re-stimulation in OVA-sensitized groups. Data expressed in terms of pg/ml culture medium. Data shown are means ± SEM of four animals/group.

* p < 0.05 vs. OVA group without OVA re-stimulation, **p < 0.01 vs. OVA group without OVA re-stimulation, #p < 0.05 vs. OVA group with OVA re-stimulation.