Article Figures & Data
Figures
- Fig. 1.
Kupffer cell CD163 cell surface marker mRNA expression in Hep:KC cultures. Hepatocytes were cultured alone (in micropatterned cocultures with 3T3-J2 fibroblasts) ± KCs (at Hep:KC of 1:0, 1:0.1, and 1:0.4). RT-PCR analysis was performed on samples from days 3, 7, 11, and 15 post-KC addition for mRNA expression of CD163. Results were relative to cultures without KCs (Hep:KC 1:0) (n = 3 ± S.D.). Mean values were significantly different from Hep:KC *1:0 and *#1:0.1 cultures (P < 0.05).
- Fig. 2.
Cytokine secretion in Hep:KC cultures after LPS treatment. Hepatocytes were cultured alone (in micropatterned cocultures with 3T3-J2 fibroblasts) ± Kupffer cells (at Hep:KC of 1:0, 1:0.1, and 1:0.4). Secreted proinflammatory cytokines (IL-1β, IL-6, IL-8, IL-10, TNF-α, and IFN-γ) were measured in cell culture media 24 hours after exposure to LPS (50 ng/ml) (n = 3 ± S.D.). Mean values were significantly different from *LPS-untreated Hep:KC cultures of matched cell ratios and *#LPS-treated 1:0 cultures (P < 0.05). aLevels were undetectable.
- Fig. 3.
Cytokine secretion in Hep:KC cultures after IL-6 and IL-1β treatment. Hepatocytes were cultured alone (in micropatterned cocultures with 3T3-J2 fibroblasts) ± KCs (at Hep:KC of 1:0, 1:0.1, and 1:0.4). Secreted proinflammatory cytokines such as IL-8, TNF-α, and IFN-γ were measured in cell culture media 4 days after exposure to IL-6 (A–C) and IL-1β (D–F) (n = 3 ± S.D.). aLevels were undetectable.
- Fig. 4.
IC50 of CYP3A4 suppression in Hep:KC cultures after IL-1β and IL-6 incubation. Hepatocytes were cultured alone (in micropatterned cocultures with 3T3-J2 fibroblasts) ± KCs (at Hep:KC of 1:0 and 1:0.4). After IL-1β and IL-6 (6.25–5000 pg/ml) incubation, CYP3A4 activity levels were measured using the CYP3A4-Glo assay at 4 days after cytokine exposure (A and C) and 6 days after recovery in cytokine-free media (B and D). IC50 values were measured using GraphPad Prism (n = 3 ± S.D.).
- Fig. 5.
Expression of cytokine receptors in Hep:KC cultures. Hepatocytes were cultured alone (in micropatterned cocultures with 3T3-J2 fibroblasts) ± KCs (at Hep:KC of 1:0, 1:0.1, and 1:0.4). RT-PCR analysis was performed on samples from days 3, 7, 11, and 15 for gene expression of (A) IL-6R, (B) IL-1R1, (C) IL-2RB, and (D) IL-23R. Results were relative to cultures without KC (Hep:KC 1:0) (n = 3 ± S.D.). Mean values were significantly different from Hep:KC *1:0 and *#1:0.1 (P < 0.05). aLevels were undetectable (Ct values for 18s housekeeping gene were consistent across all cell samples, but Ct values for IL-2RB were >35 and undetectable for IL-23R).
- Fig. 6.
Effects of IL-1 receptor antagonist (IL-1RA) cytokine and anti-IL-1β mAb (αIL-1β) on IL-1β–mediated CYP3A4 suppression. Hepatocytes were cultured alone (in micropatterned cocultures with 3T3-J2 fibroblasts) ± KCs (at Hep:KC of 1:0 and 1:0.4) and codosed with IL-1β (2.5 ng/ml or 0.1 ng/ml for Hep:KC of 1:0 or 1:0.4, respectively) and either IL-1RA (100–1000 ng/ml) or αIL-1β (10–100 ng/ml). CYP3A4 activity levels were measured using CYP3A4-Glo assay at 4 days after treatment (n = 3 ± S.D.). *Mean values were significantly different from IL-1β-untreated matched Hep:KC cultures (P < 0.05).
- Fig. 7.
Cytokine secretion in Hep:KC cultures resulting from IL-1β, IL-2, and IL-23 exposure. Hepatocytes were cultured alone (in micropatterned cocultures with 3T3-J2 fibroblasts) ± KCs (at Hep:KC of 1:0, 1:0.1, and 1:0.4). Secreted proinflammatory cytokines such as IL-8 (A), TNF-α (B), and IFN-γ (C) were measured in cell culture media 4 days after cytokine treatment (n = 3 ± S.D.). aLevels were undetectable.
- Fig. 8.
CYP3A4 activity in Hep:KC cultures after IL-2 and IL-23 treatment. Hepatocytes were cultured alone (in micropatterned cocultures with 3T3-J2 fibroblasts) ± KCs (at Hep:KC of 1:0, 1:0.1, and 1:0.4). CYP3A4 activity levels were measured after 4 days of IL-2 (A) and IL-23 (B) treatment (n = 3 ± S.D.). *Mean values were significantly different from IL-2-untreated Hep:KC cultures of matched cell ratios (P < 0.05).
- Fig. 9.
Gene expression of CYP3A4 and CRP in Hep:KC cultures after cytokine treatments. Hepatocytes were cultured (in micropatterned cocultures with 3T3-J2 fibroblasts) ± Kupffer cells (at Hep:KC of 1:0 and 1:0.4). After IL-1β (A and B) and IL-6 (C and D) incubation (6.25–5000 pg/ml), CYP3A4 and CRP mRNA levels were assessed by RT-PCR (n = 3 ± S.D.).
Tables
- TABLE 1
Effects of IL-1β and IL-6 on metabolism enzymes mRNA expression in Hep:KC (1:0 and 1:0.4) cell cultures
Metabolism Enzymes Exposure to IL-1β (6.25–5000 pg/ml), 4 Days Exposure to IL-6 (6.25–5000 pg/ml), 4 Days Effectsa KC-Dependent Effects? Effectsa KC-Dependent Effects? CYP1A1 ↔ — ↔ — CYP1A2 ↓ Yes ↓ — CYP2A6 ND — ND — CYP2B6 ↔ — ↓ — CYP2C8 ↓ Yes ↓ — CYP2C9 ↓ Yes ↓ — CYP2C9 ↔ — ↔ — CYP2C19 ND — ↔ — CYP2D6 ↔ — ↔ — CYP2E1 ↔ — ↔ — CYP3A4 ↓ Yes ↓ — GSTA1 ↓b Yes ↓ — GSTA2 ↓b Yes ↓ — SULT1A1 ND — ND — SULT1A2 ND — ND — UGT1A1 ↓ Yes ↓ — UGT2B7 ↓b Yes ↓ — - TABLE 2
Effects of IL-1β and IL-6 on uptake and efflux transporters mRNA expression in Hep:KC (1:0 and 1:0.4) cell cultures
Transporters Exposure to IL-1β (6.25–5000 pg/ml), 4 Days Exposure to IL-6 (6.25–5000 pg/ml), 4 Days Effectsa KC-Dependent Effects? Effectsa KC-Dependent Effects? NTCP ↓b Yes ↓ — OAT2 ↔ — ↔ — OATP1B1 ↓b Yes ↔ — OATP1B3 ↓b Yes ↓ — OATP2B1 ↓ — ↔ — OCT1 ↓ — ↓ — BCRP ND — ND — BSEP ↓ Yes ↓ — MDR1 ↔ — ↔ — MRP2 ↔ — ↔ — MRP3 ↔ — ↔ — MRP4 ND — ND — - TABLE 3
Effects of IL-1β and IL-6 on acute-phase proteins mRNA expression in Hep:KC (1:0 and 1:0.4) cell cultures
Proteins Exposure to IL-1β (6.25–5000 pg/ml), 4 days Exposure to IL-6 (6.25–5000 pg/ml), 4 days Effectsa (Highest Observed % Untreated Control) KC-Dependent Effects? Effects (Highest Observed % Untreated Control) KC-Dependent Effects? AAG ↑ Yes ↑ — 1250 ± 94 (1:0) 1986 ± 170 (1:0) 1459 ± 412 (1:0.4) 1024 ± 98 (1:0.4) CRP ↑ Yes ↑ — 1133 ± 260 (1:0) 95,220 ± 12,671 (1:0) 53,132 ± 15,568 (1:0.4) 80,426 ± 25,395 (1:0.4) SAA2 ↑ — ↑ — 5057 ± 1181 (1:0) 18,208 ± 6529 (1:0) 5319 ± 1688 (1:0.4) 10,392 ± 1643 (1:0.4) ↵a Effects were noted as upregulation (↑) of ≥150% of untreated control in a concentration-dependent manner.
Data Supplement
Files in this Data Supplement:
- Supplemental Data -
Supplemental Table 1 - Information of Donors Used in Pre-characterization Studies
Supplemental Figure 1 - Metabolic competency of Hep:KC cultures
Supplemental Figure 2 - Effects of IL-6 on Hep:KC cultures
Supplemental Figure 3 - Effects of IL-1-beta on Hep:KC cultures
Supplemental Figure 4 - Effects of IL-2 on Hep:KC cultures
Supplemental Figure 5 - Effects of IL-23 on Hep:KC cultures
- Supplemental Data -
In this issue
Hepatocyte-Kupffer Cell Coculture for Studying Cytokine Effects
