Selective expression and functions of interleukin 18 receptor on T helper (Th) type 1 but not Th2 cells

Abstract

Interleukin (IL)-18 induces interferon (IFN)-gamma synthesis and synergizes with IL-12 in T helper type 1 (Th1) but not Th2 cell development. We report here that IL-18 receptor (IL-18R) is selectively expressed on murine Th1 but not Th2 cells. IL-18R mRNA was expressed constitutively and consistently in long-term cultured clones, as well as on newly polarized Th1 but not Th2 cells. IL-18 sustained the expression of IL-12Rbeta2 mRNA, indicating that IL-18R transmits signals that maintain Th1 development through the IL-12R complex. In turn, IL-12 upregulated IL-18R mRNA. Antibody against an IL-18R-derived peptide bound Th1 but not Th2 clones. It also labeled polarized Th1 but not Th2 cells derived from naive ovalbumin-T cell antigen receptor-alphabeta transgenic mice (D011.10). Anti-IL-18R antibody inhibited IL-18- induced IFN-gamma production by Th1 clones in vitro. In vivo, anti-IL-18R antibody reduced local inflammation and lipopolysaccharide-induced mortality in mice. This was accompanied by shifting the balance from Th1 to Th2 responses, manifest as decreased IFN-gamma and proinflammatory cytokine production and increased IL-4 and IL-5 synthesis. Therefore, these data provide a direct mechanism for the selective effect of IL-18 on Th1 but not Th2 cells. They also show that the synergistic effect of IL-12 and IL-18 on Th1 development may be due to the reciprocal upregulation of their receptors. Furthermore, IL-18R is a cell surface marker distinguishing Th1 from Th2 cells and may be a therapeutic target.

Figure 1

Selective expression of IL-18R message in Th1 compared with Th2 cells. (a) Cloned Th1 (Dorris, X4) and Th2 cells (D10, X12) were activated with irradiated spleen cells (APCs) and antigen. RNA were extracted 5 d later and analyzed by RT-PCR Southern blot with probes of IL-18R, IL-12Rβ2, ST2L, or HPRT. (b) CD4⁺ T cells from D011.10 mice were polarized with APCs and antigen in the presence of IL-12 and anti–IL-4 antibody (Th1 line), or IL-4, anti–IL-12, and anti-IFN-γ (Th2 line). The cells were harvested 3, 5, and 7 d after start of the culture or 5 d (*5) after the second round of culture. RNA was extracted and analyzed by RT-PCR Southern blot as in a above. (c) Kinetic study of the expression of messages by representative Th1 (Dorris) and Th2 (D10) clones. Cells were stimulated with APCs and antigen, and RNA was extracted for RT-PCR Southern blot analysis on days 3, 5, and 9. For cells extracted on day 9, IL-2 was added in the culture on day 3 to maintain the viability of the cells beyond day 7. Data are representative of four experiments.

Figure 1

Selective expression of IL-18R message in Th1 compared with Th2 cells. (a) Cloned Th1 (Dorris, X4) and Th2 cells (D10, X12) were activated with irradiated spleen cells (APCs) and antigen. RNA were extracted 5 d later and analyzed by RT-PCR Southern blot with probes of IL-18R, IL-12Rβ2, ST2L, or HPRT. (b) CD4⁺ T cells from D011.10 mice were polarized with APCs and antigen in the presence of IL-12 and anti–IL-4 antibody (Th1 line), or IL-4, anti–IL-12, and anti-IFN-γ (Th2 line). The cells were harvested 3, 5, and 7 d after start of the culture or 5 d (*5) after the second round of culture. RNA was extracted and analyzed by RT-PCR Southern blot as in a above. (c) Kinetic study of the expression of messages by representative Th1 (Dorris) and Th2 (D10) clones. Cells were stimulated with APCs and antigen, and RNA was extracted for RT-PCR Southern blot analysis on days 3, 5, and 9. For cells extracted on day 9, IL-2 was added in the culture on day 3 to maintain the viability of the cells beyond day 7. Data are representative of four experiments.

Figure 1

Selective expression of IL-18R message in Th1 compared with Th2 cells. (a) Cloned Th1 (Dorris, X4) and Th2 cells (D10, X12) were activated with irradiated spleen cells (APCs) and antigen. RNA were extracted 5 d later and analyzed by RT-PCR Southern blot with probes of IL-18R, IL-12Rβ2, ST2L, or HPRT. (b) CD4⁺ T cells from D011.10 mice were polarized with APCs and antigen in the presence of IL-12 and anti–IL-4 antibody (Th1 line), or IL-4, anti–IL-12, and anti-IFN-γ (Th2 line). The cells were harvested 3, 5, and 7 d after start of the culture or 5 d (*5) after the second round of culture. RNA was extracted and analyzed by RT-PCR Southern blot as in a above. (c) Kinetic study of the expression of messages by representative Th1 (Dorris) and Th2 (D10) clones. Cells were stimulated with APCs and antigen, and RNA was extracted for RT-PCR Southern blot analysis on days 3, 5, and 9. For cells extracted on day 9, IL-2 was added in the culture on day 3 to maintain the viability of the cells beyond day 7. Data are representative of four experiments.

Figure 2

Effect of IL-18 and IL-12 on the expression of IL-18R, IL-12Rβ2, and Th1 functions. A representative Th1 clone (Dorris) was stimulated with irradiated APCs and antigen in the presence of IL-18, IL-12, a combination of IL-18 and IL-12, or medium alone. RNA was extracted on day 5 and analyzed by Northern blot. The relative level of the message was compared with reference to HPRT. Message expressed by cells cultured with medium alone was normalized as 1.0. Similar results were obtained with cells cultured for 3 or 7 d.

Figure 3

Flow cytometric analysis of cell surface expression of IL-18R. (a) Th1 (Dorris) and Th2 (D10) were stained with rabbit anti–IL-18R or preimmune serum (1/25 dilution) followed by biotinylated goat anti–rabbit IgG and were developed with PerCP-streptavidin. Similar results were obtained with X4 (Th1) and X12 (Th2) (not shown). (b) CD4⁺ T cells from OVA-TCR-αβ transgenic mice (D011.10) were driven to Th1 or Th2 lines for 6 d with APCs and antigen in the presence of IL-12 and anti–IL-4 antibody (Th1 line) or IL-4 (Th2 line). They were stained for cell surface IL-18R (with PerCP), and intracellular IFN-γ (with FITC) and IL-4 (with PE). All cells in b were activated with PMA/ionomycin for 4 h and Brefeldin A added in the last 2 h. Unfilled histograms, Staining with control preimmune serum. Similar results were obtained with cells driven for up to five rounds of culture.

Figure 3

Flow cytometric analysis of cell surface expression of IL-18R. (a) Th1 (Dorris) and Th2 (D10) were stained with rabbit anti–IL-18R or preimmune serum (1/25 dilution) followed by biotinylated goat anti–rabbit IgG and were developed with PerCP-streptavidin. Similar results were obtained with X4 (Th1) and X12 (Th2) (not shown). (b) CD4⁺ T cells from OVA-TCR-αβ transgenic mice (D011.10) were driven to Th1 or Th2 lines for 6 d with APCs and antigen in the presence of IL-12 and anti–IL-4 antibody (Th1 line) or IL-4 (Th2 line). They were stained for cell surface IL-18R (with PerCP), and intracellular IFN-γ (with FITC) and IL-4 (with PE). All cells in b were activated with PMA/ionomycin for 4 h and Brefeldin A added in the last 2 h. Unfilled histograms, Staining with control preimmune serum. Similar results were obtained with cells driven for up to five rounds of culture.

Figure 4

Effect of anti–IL-18R antibody on local inflammation. BALB/c mice were injected in the footpad with carrageenin (300 μg/50 μl/mouse). They were also injected intraperitoneally with anti–IL-18R antibody or normal rabbit IgG (0.5 mg/mouse/d) daily for 4 d, starting 24 h before the footpad injection. (a) Footpad swelling was measured daily. Data are mean ± SEM; n = 5, *P < 0.01. Footpad thickness of the antibody-treated group was indistinguishable from the control at 72 h after injection. Thus, the antibody delayed the onset of local inflammation. (b) Inflammatory score of histological examination shows extensive cellular infiltrations in the footpad of the (c) normal IgG-treated group at 24 h after carrageenin injection. This was significantly reduced after (d) anti–IL-18R injection. Sections of the carrageenin-injected footpads were stained with hematoxylin and eosin (original magnification: ×10). The number of infiltrating cells was counted and expressed as inflammatory score (0, no inflammation; 1, patchy mild inflammation; 2, patchy extensive inflammation; 3, continuous inflammation; 4, continuous inflammation with loss of architecture; n = 5, *P < 0.01). (e) DLN were pooled (five mice per group) 24 h after carrageenin injection and stimulated in vitro with immobilized anti-CD3 antibody. Supernatant was collected 48 h later, and cytokine concentration was determined by ELISA. Data are mean ± SD; n = 3, *P < 0.05, **P < 0.01. There was no significant difference in the T cell proliferative response between the two groups of mice (data not shown). Results are representative of three experiments.

Figure 4

Effect of anti–IL-18R antibody on local inflammation. BALB/c mice were injected in the footpad with carrageenin (300 μg/50 μl/mouse). They were also injected intraperitoneally with anti–IL-18R antibody or normal rabbit IgG (0.5 mg/mouse/d) daily for 4 d, starting 24 h before the footpad injection. (a) Footpad swelling was measured daily. Data are mean ± SEM; n = 5, *P < 0.01. Footpad thickness of the antibody-treated group was indistinguishable from the control at 72 h after injection. Thus, the antibody delayed the onset of local inflammation. (b) Inflammatory score of histological examination shows extensive cellular infiltrations in the footpad of the (c) normal IgG-treated group at 24 h after carrageenin injection. This was significantly reduced after (d) anti–IL-18R injection. Sections of the carrageenin-injected footpads were stained with hematoxylin and eosin (original magnification: ×10). The number of infiltrating cells was counted and expressed as inflammatory score (0, no inflammation; 1, patchy mild inflammation; 2, patchy extensive inflammation; 3, continuous inflammation; 4, continuous inflammation with loss of architecture; n = 5, *P < 0.01). (e) DLN were pooled (five mice per group) 24 h after carrageenin injection and stimulated in vitro with immobilized anti-CD3 antibody. Supernatant was collected 48 h later, and cytokine concentration was determined by ELISA. Data are mean ± SD; n = 3, *P < 0.05, **P < 0.01. There was no significant difference in the T cell proliferative response between the two groups of mice (data not shown). Results are representative of three experiments.

Figure 4

Effect of anti–IL-18R antibody on local inflammation. BALB/c mice were injected in the footpad with carrageenin (300 μg/50 μl/mouse). They were also injected intraperitoneally with anti–IL-18R antibody or normal rabbit IgG (0.5 mg/mouse/d) daily for 4 d, starting 24 h before the footpad injection. (a) Footpad swelling was measured daily. Data are mean ± SEM; n = 5, *P < 0.01. Footpad thickness of the antibody-treated group was indistinguishable from the control at 72 h after injection. Thus, the antibody delayed the onset of local inflammation. (b) Inflammatory score of histological examination shows extensive cellular infiltrations in the footpad of the (c) normal IgG-treated group at 24 h after carrageenin injection. This was significantly reduced after (d) anti–IL-18R injection. Sections of the carrageenin-injected footpads were stained with hematoxylin and eosin (original magnification: ×10). The number of infiltrating cells was counted and expressed as inflammatory score (0, no inflammation; 1, patchy mild inflammation; 2, patchy extensive inflammation; 3, continuous inflammation; 4, continuous inflammation with loss of architecture; n = 5, *P < 0.01). (e) DLN were pooled (five mice per group) 24 h after carrageenin injection and stimulated in vitro with immobilized anti-CD3 antibody. Supernatant was collected 48 h later, and cytokine concentration was determined by ELISA. Data are mean ± SD; n = 3, *P < 0.05, **P < 0.01. There was no significant difference in the T cell proliferative response between the two groups of mice (data not shown). Results are representative of three experiments.

Figure 5

Effect of anti–IL-18R antibody on LPS-induced shock. BALB/c mice were injected intraperitoneally with 18 mg/kg body wt of LPS. They were injected daily with 0.5 mg i.p. of anti–IL-18R antibody or normal rabbit IgG, starting 24 h before LPS injection. (a) Anti–IL-18R antibody treatment significantly reduced mortality compared with control (n = 20, *P < 0.001). Results are pooled from two experiments. (b) Both groups of mice suffered similar initial weight loss. However, the antibody-treated group recovered significantly more rapidly compared with control (mean body weight of surviving mice ± SEM; *P < 0.05, **P < 0.01). (c) Sera were collected and pooled 2 and 24 h after LPS injection. Cytokine concentrations in the serum were determined by ELISA. Data are mean ± SD of triplicate assays of the 2-h sera; *P < 0.05, **P < 0.01. Similar results were obtained for the 24-h sera (data not shown).