Volume 114, Issue 6, Pages 1244-1256 (June 1998) Proinflammatory cytokines differentially modulate their own expression in human intestinal mucosal mesenchymal cells  Scott A. Strong, Theresa T. Pizarro, John S. Klein, Fabio Cominelli, Claudio Fiocchi  Gastroenterology  Volume 114, Issue 6, Pages 1244-1256 (June 1998) DOI: 10.1016/S0016-5085(98)70431-7 Copyright © 1998 American Gastroenterological Association Terms and Conditions

Fig. 1 Proliferative response of human intestinal mesenchymal cells to IL-1β (1000 U/mL) and PDGF (20 ng/mL). In upper panels, HIMM proliferation was measured by incorporation of [3H]thymidine and expressed by a stimulation index; in lower panels, proliferation of the same cultures was measured as the percentage of cells in the S phase of the cell cycle as assessed by FACS analysis of nuclear staining with propidium iodide. Figure represents three separate experiments. Gastroenterology 1998 114, 1244-1256DOI: (10.1016/S0016-5085(98)70431-7) Copyright © 1998 American Gastroenterological Association Terms and Conditions

Fig. 2 Dose-dependent proliferation of human intestinal mesenchymal cells to recombinant and natural cytokines. (A) Comparative response of HIFs to increasing amounts of recombinant IL-1β (•), IL-6 (2), and TNF-α (○). (B) Comparative response of HIMMs to increasing amounts of recombinant IL-1β (•) and cell lysates adjusted to contain 0.01–1000 U/mL of natural IL-1 (○). Lysates were obtained from LPMCs isolated from IBD-affected mucosa, and the concentration of IL-1 was measured by radioimmunoassay. Proliferation was measured by incorporation of [3H]thymidine and expressed by a stimulation index. Results represent means ± SEM of five and three separate experiments for HIFs and HIMMs, respectively. Gastroenterology 1998 114, 1244-1256DOI: (10.1016/S0016-5085(98)70431-7) Copyright © 1998 American Gastroenterological Association Terms and Conditions

Fig. 3 Proliferation of human intestinal mesenchymal cells to combinations of recombinant cytokines. (A) Response of HIFs to suboptimal amounts of recombinant IL-1β, IL-6, and TNF-α (100 U/mL each), alone or in combination. (B) Response of HIMMs to suboptimal amounts of recombinant IL-1β, IL-6, and TNF-α (100 U/mL each), alone or in combination. Proliferation was measured by incorporation of [3H]thymidine and expressed by a stimulation index. Gastroenterology 1998 114, 1244-1256DOI: (10.1016/S0016-5085(98)70431-7) Copyright © 1998 American Gastroenterological Association Terms and Conditions

Fig. 4 Time-dependent induction of proinflammatory cytokine mRNA in human intestinal mesenchymal cells by recombinant IL-1β (1000 U/mL). Northern blot analysis of IL-1β, IL-6, TNF-α, and β-actin transcripts in total RNA extracted from IL-1β–, Zeta serum–, and PDGF (20 ng/mL)–stimulated HIMMs and HIFs. Figure represents three different experiments. Gastroenterology 1998 114, 1244-1256DOI: (10.1016/S0016-5085(98)70431-7) Copyright © 1998 American Gastroenterological Association Terms and Conditions

Fig. 5 Dose-dependent induction of proinflammatory cytokine mRNA in human intestinal mesenchymal cells by recombinant IL-1β (1–1000 U/mL). Northern blot analysis of IL-1β, IL-6, TNF-α, and β-actin transcripts in total RNA extracted from HIMMs and HIFs stimulated for 12 hours by IL-1β, Zeta serum, and PDGF (20 ng/mL). Figure represents three different experiments. Gastroenterology 1998 114, 1244-1256DOI: (10.1016/S0016-5085(98)70431-7) Copyright © 1998 American Gastroenterological Association Terms and Conditions

Fig. 6 Induction of cytokine protein synthesis by cytokine-stimulated human intestinal mesenchymal cells. Time-dependent secretion of immunoreactive IL-6 in culture supernatants of HIMMs (■) and HIFs (▨) after stimulation with recombinant IL-1β (100 U/mL). Gastroenterology 1998 114, 1244-1256DOI: (10.1016/S0016-5085(98)70431-7) Copyright © 1998 American Gastroenterological Association Terms and Conditions

Fig. 7 Induction of proinflammatory cytokine mRNA in human intestinal mesenchymal cells by combinations of recombinant cytokines. Northern blot analysis of IL-1β, IL-6, TNF-α, and β-actin transcripts in total RNA extracted from HIMMs and HIFs stimulated by suboptimal amounts of recombinant IL-1β, IL-6, and TNF-α (100 U/mL each), alone or in combination. Figure represents three different experiments. Gastroenterology 1998 114, 1244-1256DOI: (10.1016/S0016-5085(98)70431-7) Copyright © 1998 American Gastroenterological Association Terms and Conditions

Fig. 8 Induction of proinflammatory cytokine mRNA in human intestinal mesenchymal cells by recombinant and natural cytokines. Northern blot analysis of IL-1β, IL-6, and β-actin transcripts in total RNA extracted from HIMMs stimulated by recombinant IL-1β (1000 U/mL rIL-1β) or cell lysates adjusted to contain 1000 U/mL of natural IL-1. Lysates were obtained from Crohn's disease (CD)- and ulcerative colitis (UC)-affected LPMCs, and the concentration of immunoreactive IL-1 was measured by radioimmunoassay. Zeta serum and PDGF (20 ng/mL) served as controls. Gastroenterology 1998 114, 1244-1256DOI: (10.1016/S0016-5085(98)70431-7) Copyright © 1998 American Gastroenterological Association Terms and Conditions

Fig. 9 Dose-dependent modulation of proinflammatory cytokine mRNA in human intestinal mesenchymal cells by IL-1RA. Northern blot analysis of IL-1β, IL-6, and β-actin transcripts in total RNA extracted from Zeta serum–, IL-1RA (0.2 ng/mL)–, and IL-1β (100 U/mL) plus IL-1RA–stimulated HIMMs and HIFs. Gastroenterology 1998 114, 1244-1256DOI: (10.1016/S0016-5085(98)70431-7) Copyright © 1998 American Gastroenterological Association Terms and Conditions

Fig. 10 RT-PCR showing expression of IL-1R types in human intestinal mesenchymal cells. Total cellular RNA was extracted and reverse-transcribed, and multiplex PCR was performed using primers specific for either the human IL-1RI or IL-1RII, and for the housekeeping gene GAPDH. Amplified PCR products were resolved on a 3% agarose gel and showed that mRNA expression of IL-1RI, but not IL-1RII, is detected in control (Cont.) and Crohn's disease (CD)- and ulcerative colitis (UC)-affected HIFs and HIMMs. PBMCs cultured for 6 hours in either the presence or the absence of lipopolysaccharide (LPS; 10 μg/mL) served as positive controls. IL-1RI, 353 base pairs; IL-1RII, 508 base pairs; GAPDH, 880 base pairs. Gastroenterology 1998 114, 1244-1256DOI: (10.1016/S0016-5085(98)70431-7) Copyright © 1998 American Gastroenterological Association Terms and Conditions